Perinatal DEHP exposure modulates pituitary estrogen receptor α and ß expression altering lactotroph and somatotroph cell growth in prepuberal and adult male rats.

Phthalates are synthetic chemicals widely used to make polyvinylchloride (PVC) soft and flexible. Of these, Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used, with high human exposure occurring as early as the fetal developmental stage and affecting the endocrine system. We focused on the perinatal DEHP effects on pituitary estrogen receptor (ER) expression in male rats, explored their impact on lactotroph and somatotroph cell growth, and evaluated the direct effects of this phthalate on pituitary cell cultures. Our results showed that DEHP perinatal exposure was unable to modify the ERα+ pituitary cell number from prepuberal rats, but increased ERß+ cells. In adulthood, the pituitary ERα+ cells underwent a slight decrease with ERß showing the greatest changes, and with a significant increase observed in somatotroph cells. Also, in vitro, DEHP reduced the ERα+ cells, increased the percentage of ERß+ pituitary cells and modified the Ki67 index, as well as decreasing the lactotrophs and increasing the somatotroph cells. In conclusion, the present study showed that DEHP induced ER expression changes in normal pituitary glands from male rats in in vivo and in vitro conditions, suggesting that DEHP could differentially modulate lactotroph and somatotroph cell growth, possibly as a consequence of ER imbalance.

The phthalate DEHP modulates the estrogen receptors α and ß increasing lactotroph cell population in female pituitary glands.

Humans are exposed to numerous endocrine disruptors on a daily basis, which may interfere with endogenous estrogens, with Di-(2-ethylhexyl) phthalate (DEHP) being one of the most employed. The anterior pituitary gland is a target of 17ß-estradiol (E2) through the specific estrogen receptors (ERs) α and ß, whose expression levels fluctuate in the gland under different contexts, and the ERα/ß index is responsible for the final E2 effect. The aim of the present study was to evaluate in vivo and in vitro the DEHP effects on ERα and ß expression in the pituitary cell population, and also its impact on lactotroph and somatotroph cell growth. Our results revealed that perinatal exposure to DEHP altered the ERα and ß expression pattern in pituitary glands from prepubertal and adult female rats and increased the percentage of lactotroph cells in adulthood. In the in vitro system, DEHP down-regulated ERα and ß expression, and as a result increased the ERα/ß ratio and decreased the percentages of lactotrophs and somatotrophs expressing ERα and ß. In addition, DEHP increased the S + G2M phases, Ki67 index and cyclin D1 in vitro, leading to a rise in the lactotroph and somatotroph cell populations. These results showed that DEHP modified the pituitary ERα and ß expression in lactotrophs and somatotrophs from female rats and had an impact on the pituitary cell growth. These changes in ER expression may be a mechanism underlying DEHP exposure in the pituitary gland, leading to cell growth deregulation.

Oxidative stress and mitochondrial adaptive shift during pituitary tumoral growth.

The cellular transformation of normal functional cells to neoplastic ones implies alterations in the cellular metabolism and mitochondrial function in order to provide the bioenergetics and growth requirements for tumour growth progression. Currently, the mitochondrial physiology and dynamic shift during pituitary tumour development are not well understood. Pituitary tumours present endocrine neoplastic benign growth which, in previous reports, we had shown that in addition to increased proliferation, these tumours were also characterized by cellular senescence signs with no indication of apoptosis. Here, we show clear evidence of oxidative stress in pituitary cells, accompanied by bigger and round mitochondria during tumour development, associated with augmented biogenesis and an increased fusion process. An activation of the Nrf2 stress response pathway together with the attenuation of the oxidative damage signs occurring during tumour development were also observed which will probably provide survival advantages to the pituitary cells. These neoplasms also presented a progressive increase in lactate production, suggesting a metabolic shift towards glycolysis metabolism. These findings might imply an oxidative stress state that could impact on the pathogenesis of pituitary tumours. These data may also reflect that pituitary cells can modulate their metabolism to adapt to different energy requirements and signalling events in a pathophysiological situation to obtain protection from damage and enhance their survival chances. Thus, we suggest that mitochondria function, oxidative stress or damage might play a critical role in pituitary tumour progression.

Estrogen receptor ß regulates the tumoral suppressor PTEN to modulate pituitary cell growth.

In this study, we focused on ERß regulation in the adenohypophysis under different estrogenic milieu, by analyzing whether ER modulates the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression and its subcellular localization on anterior pituitary glands from Wistar rats and GH3 lactosomatotroph cells that over-expressed ERß. ERß was regulated in a cyclic manner, and underwent dynamic changes throughout the estrous cycle, with decreased ERß+ cells in estrus and under E2 treatment, but increased in ovariectomized rats. In addition, the ERα/ß ratio increased in estrus and under E2 stimulation, but decreased in ovariectomized rats. Double immunofluorescence revealed that lactotroph and somatotroph ERß+ were significantly decreased in estrus. Also, variations in the PTEN expression was observed, which was diminished with high E2 conditions but augmented with low E2 milieu. The subcellular localization of this phosphatase was cell cycle-dependent, with remarkable changes in the immunostaining pattern: nuclear in arrested pituitary cells but cytoplasmic in stimulated cells, and responding differently to ER agonists, with only DPN being able to increase PTEN expression and retaining it in the nucleus. Finally, ERß over-expression increased PTEN with a noticeable subcellular redistribution, and with a significant nuclear signal increase in correlation with an increase of cells in G0/G1 phase. These results showed that E2 is able to inhibit ERß expression and suggests that the tumoral suppressor PTEN might be one of the signaling proteins by which E2, through ERß, acts to modulate pituitary cell proliferation, thereby adapting endocrine populations in relation with hormonal necessities.

Inhibitory role of ERß on anterior pituitary cell proliferation by controlling the expression of proteins related to cell cycle progression.

Considering that the role of ERß in the growth of pituitary cells is not well known, the aim of this work was to determine the expression of ERß in normal and tumoral cells and to investigate its implications in the proliferative control of this endocrine gland, by analyzing the participation of cyclin D1, Cdk4 and p21. Our results showed that the expression of ERß decreased during pituitary tumoral development induced by chronic E2 stimulation. The 20 ± 1.6% of normal adenohypophyseal cells expressed ERß, with this protein being reduced in the hyperplastic/adenomatous pituitary: at 20 days the ERß+ population was 10.7 ± 2.2%, while after 40 and 60 days of treatment an almost complete loss in the ERß expression was observed (40 d: 1 ± 0.6%; 60 d: 2 ± 0.6%). The ERα/ß ratio increased starting from tumors at 40 days, mainly due to the loss of ERß expression. The cell proliferation was analyzed in normal and hyperplastic pituitary and also in GH3ß- and GH3ß+ which contained different levels of ERß expression, and therefore different ERα/ß ratios. The over-expression of ERß inhibited the GH3 cell proliferation and expression of cyclin D1 and ERα. Also, the ERß activation by its agonist DPN changed the subcellular localization of p21, inducing an increase in the p21 nuclear expression, where it acts as a tumoral suppressor. These results show that ERß exerts an inhibitory role on pituitary cell proliferation, and that this effect may be partially due to the modulation of some key regulators of the cell cycle, such as cyclin D1 and p21. These data contribute significantly to the understanding of the ER effects in the proliferative control of pituitary gland, specifically related to the ERß function in the E2 actions on this endocrine gland.

Glutamate release machinery is altered in the frontal cortex of rats with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model that mimics many of the clinical and pathological features of the human disease multiple sclerosis (MS). Both are inflammatory demyelinating and neurodegenerative pathologies of the central nervous system associated with motor, sensory, and cognitive deficits. In MS, gray matter atrophy is related to the emergence of cognitive deficits and contributes to clinical progression. In particular, prefrontal cortex injury and dysfunction have been correlated to the development of fatigue, one of the most common and disabling symptoms in MS. However, the molecular bases of these changes remain unknown. Taking advantage of EAE similitude, we herein analyze functional and morphological changes in isolated cortical presynaptic terminals (synaptosomes) from an acute rat model. We found impaired glutamate release in the frontal cortex from EAE rats. This defect appeared along with the onset of the disease, reversing when clinical signs were no more evident. Biochemical analysis of EAE synaptosomes revealed alterations in the presynaptic release machinery and in the response to depolarization, which was accompanied by abnormal synapsin I phosphorylation and dispersion. These changes were associated with reduced synaptic vesicle mobility, with no alterations in synaptosomal morphology as evidenced by electron microscopy. The present are the first pieces of evidence unraveling the molecular mechanisms of frontal cortex neuronal dysfunction in EAE and, possibly, MS.

Cooperative effect of E2 and FGF2 on lactotroph proliferation triggered by signaling initiated at the plasma membrane.

In the present work, we investigated the effect of 17ß-estradiol (E2) and basic fibroblast growth factor 2 (FGF2) on the lactotroph cell-proliferative response and the related membrane-initiated signaling pathway. Anterior pituitary mixed-cell cultures of random, cycling 3-mo-old female rats were treated with 10 nM E2, E2 membrane-impermeable conjugated BSA (E2-BSA), PPT (ERα agonist), and DPN (ERß agonist) alone or combined with FGF2 (10 ng/ml) for 30 min or 4 h. Although our results showed that the uptake of BrdU into the nucleus of lactotrophs was not modified by E2 or FGF2 alone, a significant increase in the lactotroph uptake of BrdU was observed after E2/FGF2 coincubation, with this effect being mimicked by PPT/FGF2. These proliferative effects were blocked by ICI 182,780 or PD-98059. The involvement of membrane ER in the proliferative response of prolactin cells induced by the steroid and FGF2 coincubation was confirmed using E2-BSA, and the association between ERα and FGF receptor was observed after E2/FGF2 treatment by immunoprecipitation. A significant increase in the ERK1/2 expression was noted after E2, E2-BSA, PPT, and FGF2 alone, which was more noticeable after E2-BSA/FGF2, E2/FGF2, or PPT/FGF2 treatments. This study provides evidence that E2 and FGF2 exert a cooperative effect on the lactotroph proliferation principally by signaling initiated at the plasma membrane triggering a genomic effect mediated by MEK/ERK1/2, a common signaling pathway, that finally regulates the lactotroph population, thus contributing to pituitary plasticity.

17ß-Estradiol stimulates the translocation of endogenous estrogen receptor α at the plasma membrane of normal anterior pituitary cells.

In the present work we aimed at identifying ERα in the plasma membrane of normal anterior pituitary cells and investigated if 17ß-estradiol was able to induce their subcellular redistribution. Our results show that about 8% of anterior pituitary cells expressed ERα in the plasma membrane, with the geometrical mean fluorescence intensity being increased after steroid hormone treatment. 17ß-Estradiol and the selective ERα agonist PPT induced an increase of ERα expression in the plasma membrane and activated the PKCα/ERK 1/2 pathway in a time-course not compatible with genomic actions, thus supporting the notion of membrane-initiated effects. These findings suggest that 17ß-estradiol stimulates the translocation of endogenous ERα to the plasma membrane, consequently modulating this ER pool and leading to cellular biological effects in normal anterior pituitary gland.

17ß-Estradiol modulates the prolactin secretion induced by TRH through membrane estrogen receptors via PI3K/Akt in female rat anterior pituitary cell culture.

Considering that estradiol is a major modulator of prolactin (PRL) secretion, the aim of the present study was to analyze the role of membrane estradiol receptor-α (mERα) in the regulatory effect of this hormone on the PRL secretion induced by thyrotropin-releasing hormone (TRH) by focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway activation. Anterior pituitary cell cultures from female rats were treated with 17ß-estradiol (E(2), 10 nM) and its membrane-impermeable conjugated estradiol (E(2)-BSA, 10 nM) alone or coincubated with TRH (10 nM) for 30 min, with PRL levels being determined by RIA. Although E(2), E(2)-BSA, TRH, and E(2)/TRH differentially increased the PRL secretion, the highest levels were achieved with E(2)-BSA/TRH. ICI-182,780 did not modify the TRH-induced PRL release but significantly inhibited the PRL secretion promoted by E(2) or E(2)-BSA alone or in coincubation with TRH. The PI3K inhibitors LY-294002 and wortmannin partially inhibited the PRL release induced by E(2)-BSA, TRH, and E(2)/TRH and totally inhibited the PRL levels stimulated by E(2)-BSA/TRH, suggesting that the mER mediated the cooperative effect of E(2) on TRH-induced PRL release through the PI3K pathway. Also, the involvement of this kinase was supported by the translocation of its regulatory subunit p85α from the cytoplasm to the plasma membrane in the lactotroph cells treated with E(2)-BSA and TRH alone or in coincubation. A significant increase of phosphorylated Akt was induced by E(2)-BSA/TRH. Finally, the changes of ERα expression in the plasmalemma of pituitary cells were examined by confocal microscopy and flow cytometry, which revealed that the mobilization of intracellular ERα to the plasma membrane of lactotroph cells was only induced by E(2). These finding showed that E(2) may act as a modulator of the secretory response of lactotrophs induced by TRH through mER, with the contribution by PI3K/Akt pathway activation providing a new insight into the mechanisms underlying the nongenomic action of E(2) in the pituitary.

Soluble p-cadherin found in human semen.

Cadherins constitute a family of calcium-dependent cell-cell adhesion molecules. P (placental)-cadherin is a 118-kd protein expressed by basal cells in epithelial tissues. P-cadherin also has been described as a soluble protein in certain biological fluids, including human serum and breast milk. Here, we report the presence of an 80-kD fragment of P-cadherin in human semen. No significant differences were found in semen samples from fertile and nonfertile patients. Our results add evidence to previous data indicating that soluble fragments of P-cadherin have a widespread distribution in bodily fluids and suggest that soluble P-cadherin might have functions other than basal epithelial cell-cell adhesion.