Tibolone regulates systemic metabolism and the expression of sex hormone receptors in the central nervous system of ovariectomised rats fed with high-fat and high-fructose diet.

Estrogen replacement therapy decreases some risk factors of the metabolic syndrome but increases the risk of some types of cancer. Tibolone (TIB) has shown similar neuroprotective effects as estrogens. This study aimed to evaluate the effects of TIB on metabolic parameters and the expression of sex hormone receptors in the CNS in ovariectomised rats fed with a hypercaloric diet. Sprague-Dawley female rats were ovariectomised and fed for 30 days with a standard diet (SD) or high-fat high-fructose diet (HFFD) and treated with TIB (1 mg/kg) or vehicle. At the end of the treatments, HFFD increased body weight, glucose tolerance, triglycerides and cholesterol levels, while TIB treatment decreased these parameters. Subsequently, the hippocampus, the hypothalamus and the frontal cortex were dissected. RT-PCR was performed for progesterone receptor (PR), androgen receptor (AR), estrogen receptors alpha and beta (ERα, ERß), insulin receptor (IR) and insulin-like growth factor 1 (IGF-1). HFFD altered the expression of sex hormone receptors in specific brain structures involved in the regulation of homeostasis and cognition, which highlights the importance of a healthy diet. In turn, TIB modulated the expression of these receptors, particularly in the hypothalamus.

Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line.

Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12 h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6 h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12 h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells.

Effects of RU486 in the expression of progesterone receptor isoforms in the hypothalamus and the preoptic area of the rat during postpartum estrus.

In several mammalian species females undergo postpartum estrus, a brief period of ovulation and sexual receptivity that in rats usually occurs during the first 24h following parturition. The maximal lordotic expression occurs at 12h after the initiation of parturition and depends on intracellular progesterone receptor (PR). We studied the regulation of PR expression by its antagonist, RU486 in the hypothalamus and the preoptic area of the rat during postpartum estrus by Western blot. Adult female rats were treated with RU486 (1.25 and 5mg) 3h after parturition, and Western blot was performed to assess the expression of PR-A and PR-B at 12h postpartum. RU486 (1.25 and 5mg) reduced the expression of PR-A (63% and 95%) and that of PR-B (75% and 99%), respectively in the preoptic area whereas it had no effects in the hypothalamus. These results suggest a differential regulation of PR expression in the rat brain during postpartum estrus.

Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus.

Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 µg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 µg/µl (4 nmol) of PR-B and total PR (PR-A+PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A+B antisense (PR A+B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A+B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A+B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the regulation of the content of TPH, TH and GAD in the rat hypothalamus.

Role of progesterone receptors during postpartum estrus in rats.

We studied the role of progesterone receptor (PR) in the display of female sexual behavior during postpartum estrus in rats. Adult female rats were treated with the PR antagonist, RU486 (1.25 and 5 mg), 3 h after parturition and sexual behavior was evaluated throughout the first postpartum day. Estradiol and progesterone serum levels changed during the first 24 h postpartum. The highest estradiol and progesterone levels were found at 9 and 12 h postpartum, respectively. The predominant PR isoform in the hypothalamus and the preoptic area was PR-A during postpartum day. The content of PR-A increased at 6 h postpartum in the hypothalamus and the preoptic area, and decreased in both regions at 9 h. PR-B content only increased in the preoptic area at 12 h postpartum. The highest display of lordotic and proceptive behaviors were found at 12 h postpartum. The treatment with 1.25 and 5 mg of RU486 respectively reduced lordosis by 61% and 92% at 12 h postpartum. These results suggest that PR is essential in the display of postpartum estrus in rats.

Effects of short-term hormonal replacement on learning and on basal forebrain ChAT and TrkA content in ovariectomized rats.

It has been proposed that sex steroid hormones improve performance in some cognitive tasks by regulating the basal forebrain cholinergic function. However, the molecular basis of such influence still remains unknown. Current study analyzed the performance of ovariectomized rats in an autoshaping learning task after a short-term treatment with 17ß-estradiol (E2: 4 and 40µg/kg) and/or progesterone (P4: 4mg/kg). These results were correlated with basal forebrain choline acetyltransferase (ChAT) and TrkA protein content. The high dose of E2 enhanced both acquisition in the autoshaping task and the content of ChAT and TrkA. P4 treatment increased ChAT and TrkA content without affecting performance of rats in the autoshaping learning task. Interestingly, the continuous and simultaneous administration of E2 plus P4 did not significantly modify behavioral and biochemical evaluated parameters. These results address the influence of both E2 and P4 on cholinergic and TrkA activity and suggest that the effects of ovarian hormones on cognitive performance involve basal forebrain cholinergic neurons.

Progesterone receptor immunoreactivity differs in the uterus of pseudopregnant and medroxyprogesterone acetate-treated rabbits.

Progesterone receptor (PR) plays an important role in mammals pregnancy which is characterized by greater progesterone plasma concentrations. We assessed PR protein distribution in the rabbit uterus by immunohistochemistry in two progestational conditions: pseudopregnancy (intact adult animals treated with hCG) and after application of a synthetic progestin, medroxyprogesterone acetate (MPA), to ovariectomized animals (OVX). PR immunoreactivity in uterine epithelium of pseudopregnant rabbits was increased in relation to non-pseudopregnant (NP) rabbits. Amounts were similar on Days 1, 3, and 5 of treatment, and was greater on Day 7 (P<0.001). In contrast, a significant diminution in PR immunoreactivity was observed in stroma cells from Days 1 to 7 (P<0.001). In OVX rabbits treated with MPA, an increase in PR immunoreactivity was observed in the uterine epithelium on Days 1 to 5 of treatment, reaching a maximum on Day 3 (P<0.001). In contrast, in stromal cells a diminution in PR immunoreactivity was observed when compared to the OVX group on Days 1, 3 and 7 of MPA treatment (P<0.001), and there was a slight increase on Day 5. Results suggest a differential time course and tissue specific immunoreactivity for PR in the uterus of the rabbit in two progestational conditions. The present study indicated synthetic progestins have different mechanisms of receptor regulation than those of natural hormones and it should be taken into account in reproductive applications.

Effects of mating on progesterone receptor isoforms in rat hypothalamus.

In rodents, the display of sexual behavior during proestrus-estrus transition depends on the effect of estradiol and progesterone. Progesterone exerts its effects through intracellular receptor (PR) of which two isoforms (PR-A and PR-B) are found, with different regulation and function. In this study the effects of mating on the expression pattern of PR isoforms in the hypothalamus were investigated during proestrus-estrus transition by using western blot. PR-B isoform content significantly diminished during proestrus-estrus transition both in mated and nonmated female rats. In contrast, PR-A isoform content significantly increases during this period in nonmated rats, whereas it does not change in mated animals. These data show that PR isoforms are differentially expressed throughout proestrus-estrus transition and that mating modifies PR isoforms expression in the hypothalamus of the rat.

Role of progesterone receptor isoforms in female sexual behavior induced by progestins in rats.

Progesterone and its ring A reduced metabolites regulate female sexual behavior through the direct or indirect activation of progesterone receptor (PR) which has two isoforms with different function and regulation: PR-A and PR-B. The contribution of each PR isoform to the regulation of lordosis in rats is unknown. We explored the role of PR isoforms in lordosis display induced by progesterone and two of its ring A reduced metabolites: 5alpha-pregnan-3,20-dione (5alpha-DHP), and 5beta,3beta-pregnan-20-one (5beta,3beta-Pgl) in adult ovariectomized rats. Two weeks after ovariectomy, the animals were injected subcutaneously with 5 microg of estradiol benzoate (EB), and 40 h later, progestins were injected intracerebroventricularly. PR-B and total PR (PR-A + PR-B) sense or antisense oligonucleotides were administered intracerebroventricularly immediately before EB injection and 24 h later. Lordosis was evaluated 30, 120 and 240 min after progestin administration. Western blot analysis of both PR isoforms was performed in the hypothalamus and preoptic area 24 h after lordosis tests. All progestins induced maximal lordosis 120 min after administration, and antisense oligonucleotides against both PR isoforms inhibited lordosis in all animals. PR-B antisense oligonucleotides also inhibited lordosis induced by progesterone and 5alpha-DHP although with less efficacy than total PR antisense oligonucleotides, but the former inhibited lordosis induced by 5beta,3beta-Pgl in a similar manner as total PR antisense oligonucleotides. In the hypothalamus and preoptic area, the content of both PR isoforms or PR-B alone was diminished by the administration of total or PR-B antisense oligonucleotides, respectively. These results suggest that the PR-B isoform is essential for the display of the lordosis behavior in rats.

Biotin deficiency and biotin excess: effects on the female reproductive system.

Biotin deficiency and biotin excess have both been found to affect reproduction and cause teratogenic effects. In the reproductive tract, however, the effects of biotin have not been well established yet. We investigated the effects of varying biotin content diets on the oestrus cycle, ovarian morphology, estradiol and progesterone serum levels, and the uterine mRNA abundance of their nuclear receptors, as well as on the activity of the estradiol-degrading group of enzymes cytochrome P450 (CYP) in the liver. Three-week-old female BALB/cAnN Hsd mice were fed a biotin-deficient, a biotin-control, or a biotin-supplemented diet (0, 7.2 or 400 micromol of free biotin/kg diet, respectively) over a period of nine weeks. Striking effects were observed in the biotin-deficient group: mice showed arrested estrous cycle on the day of diestrus and changes in ovary morphology. Estradiol serum concentration increased 49.2% in biotin-deficient mice compared to the control group, while the enzymatic activities of CYP1A2 and CYP2B2 increased (P<0.05). The mRNA abundance of nuclear estrogen and progesterone receptors decreased in the biotin-deficient mice. In the biotin-supplemented group we found that, in spite of a significant (P<0.05) decrease in the number of primary and Graafian follicles and in CYP1A2 activities, mice exhibited 105.4% higher serum estradiol concentration than the control group. No changes in the expression of the nuclear receptors were observed. No significant differences were observed in serum progesterone among the groups. Our results indicate that both the deficiency and the excess of biotin have significant effects on the female mouse reproductive system.

Effects of progesterone and its reduced metabolites, dihydroprogesterone and tetrahydroprogesterone, on the expression and phosphorylation of glycogen synthase kinase-3 and the microtubule-associated protein tau in the rat cerebellum.

Progesterone exerts a variety of actions in the brain, where it is rapidly metabolized to 5alpha-dihydroprogesterone (DHP) and 3alpha,5alpha-tetrahydroprogesterone (THP). The effect of progesterone and its metabolites on the expression and phosphorylation of the microtubule-associated protein Tau and glycogen synthase kinase 3beta (GSK3beta), a kinase involved in Tau phosphorylation, were assessed in two progesterone-sensitive brain areas: the hypothalamus and the cerebellum. Administration of progesterone, DHP, and THP to ovariectomized rats did not affect Tau and GSK3beta assessed in whole hypothalamic homogenates. In contrast, progesterone and its metabolites resulted in a significant decrease in the expression of Tau and GSK3beta in the cerebellum. Furthermore, progesterone administration resulted in an increase in the phosphorylation of two epitopes of Tau (Tau-1 and PHF-1) phosphorylated by GSK3beta, but did not affect the phosphorylation of an epitope of Tau (Ser262) that is GSK3beta insensitive. These effects were accompanied by a decrease in the phosphorylation of GSK3beta in serine, which is associated to an increase in its activity, suggesting that the effect of progesterone on Tau-1 and PHF-1 phosphorylation in the cerebellum is mediated by GSK3beta. The regulation of Tau expression and phosphorylation by progesterone may contribute to the hormonal regulation of cerebellar function by the modification of neuronal cytoskeleton.

Effects of estradiol and progesterone on gastric mucosal response to early Helicobacter pylori infection in female gerbils.

BACKGROUND: Gender differences have been shown regarding the changes in the inflammatory response, gastrin secretion, and gastric acidity during Helicobacter pylori infection. AIM: To investigate the role of estradiol and progesterone in the changes of the gastric mucosa induced by H. pylori during the early stage of infection in female gerbils. MATERIALS AND METHODS: Thirty-three adult ovariectomized female gerbils were infected with H. pylori (SS1); 7 days after infection they were treated with low and high doses of estradiol (50 and 250 microg/60 days pellet), progesterone (15 and 50 mg/60 days pellet) and vehicle. Non-ovariectomized infected gerbils were used as control. Gerbils were euthanized after 6 weeks of infection. Histologic evaluation, immunohistochemical detection of proliferation cell nuclear antigen (PCNA), gastrin, and apoptosis by terminal deoxynucleotide nick end labeling (TUNEL) assay were performed. Positive cells for PCNA, TUNEL, and gastrin were counted in 10 oriented glands per animal. Two-sided p = .05 was considered significant. RESULTS: Estradiol-treated groups showed more intense and extended acute and follicular gastritis compared to the vehicle group, whereas progesterone-treated groups presented less gastritis than the other groups. Proliferation and apoptosis indexes were significantly lower in the vehicle group when compared with those of the control; both indexes were increased in the high-dose estradiol and progesterone groups as compared with those of the vehicle. Grade I nonmetaplastic atrophy was observed in the vehicle and progesterone groups. The high-dose progesterone group showed a significant reduction in the number of gastrin cells. CONCLUSIONS: Estradiol and progesterone participate in the gastric mucosal response to early H. pylori infection in gerbils.

Changes in the content of steroid receptor coactivator-1 and silencing mediator for retinoid and thyroid hormone receptors in the rat brain during the estrous cycle.

In this work, we determined the variations in the content of the steroid receptor coactivator (SRC-1) and the silencing mediator for retinoic acid and thyroid hormone receptors corepressor (SMRT) in the hypothalamus, the preoptic area, and the hippocampus of adult intact rats during the estrous cycle by Western blot. SRC-1 content changed only in the hypothalamus where its lowest content was found on diestrus day with a significant increase at proestrus. This increase was maintained on estrus day. In contrast, SMRT content changed only in the preoptic area where it diminished at metestrus in comparison with the other days of the cycle. SRC-1 content was higher than that of SMRT in the hypothalamus throughout the estrous cycle, whereas SMRT content was higher in the preoptic area. In the hippocampus, there were no significant differences in the content of any cofactor. These results demonstrate that SRC-1 and SMRT content change in a tissue-specific manner in the rat brain during the estrous cycle, and suggest that the transcriptional activity of steroid hormone receptors in the rat brain in physiological conditions is regulated by changes in SRC-1 and SMRT content.

The 26S proteasome participates in the sequential inhibition of estrous behavior induced by progesterone in rats.

Estrous behavior induced by progesterone (P) treatment of estradiol-primed rats is followed by a period in which females do not respond behaviorally to a second administration of P [sequential inhibition (SI)]. SI is thought to involve P-dependent down-regulation of hypothalamic P receptor (PR) content. This study tested the hypothesis that the 26S proteasome participates in the regulation of SI and brain PR content in female rats. Ovariectomized, estrogen-primed (estradiol benzoate, 2 microg s.c.) adult rats were injected with P (1 mg s.c.) alone or P with the proteasome inhibitors Z-Ile-Glu (OBu(1))-Ala-Leu-H (PSI, 300 microg/100 g s.c.) or N alpha-tosyl-lysyl chloromethyl ketone (TLCK, 200 microg i.p.) administered 48 h after estradiol priming. Sexual behavior was assessed in all animals 4 h later. These two agents inhibit 26S proteasome-mediated protein degradation by different mechanisms. To explore SI, the animals received a second P injection 24 h after the first, and a second sexual behavior test was performed 4 h later. After this test, brains were excised, and proteins were extracted from the preoptic area and the hypothalamus and processed for semiquantitative immunoblotting. In the first sexual behavior test (facilitation test), all animals treated with estradiol + P exhibited intense lordosis behavior. In the second sexual behavior test (inhibition test), both lordosis and proceptivity were significantly reduced in response to the second administration of P (SI). The magnitude of SI was significantly attenuated by the administration of either PSI or TLCK concurrently with the first P injection. The first P injection reduced PR content in the hypothalamus but not in the preoptic area. In contrast, PSI and TLCK significantly increased PR content in both structures. Our results suggest that PR degradation by the 26S proteasome participates in the expression of P-induced SI in female rats.

La progesterona y sus metabolitos en el funcionamiento del sistema nervioso central / Progesterone and its metabolites the functions of the central nervous system

La progesterona (P4) y sus metabolitos participan en distintas funciones del sistema nervioso central (SNC) entre las que destacan la excitabilidad neuronal, la reproducción, y las conductas asociadas a ésta. P4 y sus metabolitos actúan en las neuronas y en las células gliales a través de su interacción con: 1) receptores intracelulares específicos; 2) sitio de regulación presentes en los receptores a neurotransmisores; y 3) canales iónicos. Por estos mecanismos se inducen cambios en la expresión de genes específicos, la formación de segundos mensajeros y la conductancia iónica. La mayoría de las acciones de los metabolitos de P4 en el SNC, ocurren a nivel membranal, mientras que las de P4 son principalmente a nivel nuclear y están mediadas por la activación de los receptores intracelulares. Así, P4 y sus metabolitos pueden modificar el funcionamiento de distintas regiones del SNC, a corto (milisegundos), mediano (minutos) y largo plazo (horas y días). El conocimiento de los mecanismos moleculares por los cuales P4 y sus metabolitos participaron en el funcionamiento del SNC, permitirá entender procesos biológicos fundamentales como la conducta sexual y la reproducción; además, contribuirá al diseño de terapias alternativas en el tratamiento de diferentes trastornos neurológicos y psiquiátricos como la epilepsia, la ansiedad, el síndrome premenstrual y algunos tumores cerebrales que tienen regulación hormonal