Copper in Gynecological Diseases.

Copper (Cu) is an essential micronutrient for the correct development of eukaryotic organisms. This metal plays a key role in many cellular and physiological activities, including enzymatic activity, oxygen transport, and cell signaling. Although the redox activity of Cu is crucial for enzymatic reactions, this property also makes it potentially toxic when found at high levels. Due to this dual action of Cu, highly regulated mechanisms are necessary to prevent both the deficiency and the accumulation of this metal since its dyshomeostasis may favor the development of multiple diseases, such as Menkes' and Wilson's diseases, neurodegenerative diseases, diabetes mellitus, and cancer. As the relationship between Cu and cancer has been the most studied, we analyze how this metal can affect three fundamental processes for tumor progression: cell proliferation, angiogenesis, and metastasis. Gynecological diseases are characterized by high prevalence, morbidity, and mortality, depending on the case, and mainly include benign and malignant tumors. The cellular processes that promote their progression are affected by Cu, and the mechanisms that occur may be similar. We analyze the crosstalk between Cu deregulation and gynecological diseases, focusing on therapeutic strategies derived from this metal.

Interplay between nitric oxide and gonadotrophin-releasing hormone in the neuromodulation of the corpus luteum during late pregnancy in the rat.

BACKGROUND: Nitric oxide and GnRH are biological factors that participate in the regulation of reproductive functions. To our knowledge, there are no studies that link NO and GnRH in the sympathetic ganglia. Thus, the aim of the present work was to investigate the influence of NO on GnRH release from the coeliac ganglion and its effect on luteal regression at the end of pregnancy in the rat. METHODS: The ex vivo system composed by the coeliac ganglion, the superior ovarian nerve, and the ovary of rats on day 21 of pregnancy was incubated for 180 min with the addition, into the ganglionic compartment, of L-NG-nitro arginine methyl ester (L-NAME), a non-selective NO synthase inhibitor. The control group consisted in untreated organ systems. RESULTS: The addition of L-NAME in the coeliac ganglion compartment decreased NO as well as GnRH release from the coeliac ganglion. In the ovarian compartment, and with respect to the control group, we observed a reduced release of GnRH, NO, and noradrenaline, but an increased production of progesterone, estradiol, and expression of their limiting biosynthetic enzymes, 3ß-HSD and P450 aromatase, respectively. The inhibition of NO production by L-NAME in the coeliac ganglion compartment also reduced luteal apoptosis, lipid peroxidation, and nitrotyrosine, whereas it increased the total antioxidant capacity within the corpora lutea. CONCLUSION: Collectively, the results indicate that NO production by the coeliac ganglion modulates the physiology of the ovary and luteal regression during late pregnancy in rats.

Neuromodulatory effect of GnRH from coeliac ganglion on luteal regression in the late pregnant rat.

The GnRH/GnRH receptor system has been found in several extrapituitary tissues, although its physiological significance has not yet been well established. Taking into account that the peripheral neural system can act as a modulator of pregnancy corpus luteum, the objective was to physiologically investigate the presence of the GnRH system in coeliac ganglion (CG) and to analyse its possible involvement in luteal regression through the superior ovarian nerve (SON) at the end of pregnancy in the rat. The integrated ex vivo CG-SON-Ovary system of rats on day 21 of pregnancy was used. Cetrorelix (CTX), a GnRH receptor antagonist, was added into the ganglionic compartment while the control systems were untreated. Ganglionic GnRH release was detected under basal conditions. Then, the CTX addition in CG increased it, which would indicate the blockade of the receptor. In turn, CTX in CG caused an increase in ovarian progesterone release. Furthermore, the luteal cells showed an increase in the expression of Hsd3b1 and a decrease in the expression of Akr1c3 (progesterone synthesis and degradation enzymes, respectively), reduced TUNEL staining according to an increase in the antioxidant defence system activity and low lipid peroxide levels. The ovarian and ganglionic nitric oxide (NO) release increased, while the luteal nitrotyrosine content, measured as nitrosative stress marker, decreased. CTX in CG decreased the ovarian noradrenaline release. The present study provides evidence that GnRH from CG may trigger neuronal signals that promote the luteal regression in late pregnancy by affecting the release of NO and noradrenaline in the ovary.

The production of nitric oxide in the coeliac ganglion modulates the effect of cholinergic neurotransmission on the rat ovary during the preovulatory period.

The aim of the present work was to investigate whether the nitric oxide produced by the nitric oxide/nitric oxide synthase (NO/NOS) system present in the coeliac ganglion modulates the effects of cholinergic innervation on oxidative status, steroidogenesis and apoptotic mechanisms that take place in the rat ovary during the first proestrous. An ex vivo Coeliac Ganglion- Superior Ovarian Nerve- Ovary (CG-SON-O) system was used. Cholinergic stimulation of the CG was achieved by 10-6 M Acetylcholine (Ach). Furthermore, 400 µM Aminoguanidine (AG) - an inhibitor of inducible-NOS was added in the CG compartment in absence and presence of Ach. It was found that Ach in the CG compartment promotes apoptosis in ovarian tissue, probably due to the oxidative stress generated. AG in the CG compartment decreases the release of NO and progesterone, and increases the release of estradiol from the ovary. The CG co-treatment with Ach and AG counteracts the effects of the ganglionic cholinergic agonist on ovarian oxidative stress, increases hormone production and decreases Fas mRNA expression. These results suggest that NO is an endogenous modulator of cholinergic neurotransmission in CG, with implication in ovarian steroidogenesis and the apoptotic mechanisms that take place in the ovary during the preovulatory period in rats.

Circadian rhythms of factors involved in luteal regression are modified in p55 tumour necrosis factor receptor (TNFRp55)-deficient mice.

The rhythm of factors involved in luteal regression is crucial in determining the physiological duration of the oestrous cycle. Given the role of tumour necrosis factor (TNF)-α in luteal function and circadian regulation and that most of the effects of TNF-α are mediated by p55 TNF receptor (TNFRp55), the aims of the present study were to analyse the following during the luteal regression phase in the ovary of mice: (1) whether the pattern of expression of progesterone (P4) and the enzymes involved in the synthesis and degradation of P4 is circadian and endogenous (the rhythm persists in constant conditions, (i.e., constant darkness) with a period of about 24 hours); (2) circadian oscillations in clock gene expression; (3) whether there are daily variations in the expression of key genes involved in apoptosis and antioxidant mechanisms; and (4) the consequences of TNFRp55 deficiency. P4 was found to oscillate circadianally following endogenous rhythms of clock factors. Of note, TNFRp55 deficiency modified the circadian oscillation in P4 concentrations and its enzymes involved in the synthesis and degradation of P4, probably as a consequence of changes in the circadian oscillations of brain and muscle ARNT-Like protein 1 (Bmal1) and Cryptochrome 1 (Cry1). Furthermore, TNFRp55 deficiency modified the circadian rhythms of apoptosis genes, as well as antioxidant enzymes and peroxidation levels in the ovary in dioestrus. The findings of the present study strengthen the hypothesis that dysregulation of TNF-α signalling may be a potential cause for altered circadian and menstrual cycling in some gynaecological diseases.

Neuromodulatory effect of oestradiol in the metabolism of ovarian progesterone and oestradiol during dioestrus II: participation of the superior mesenteric ganglion.

The aims of the present study were to determine: (1) whether oestradiol (E2) in the superior mesenteric ganglion (SMG) modifies the release of ovarian progesterone (P4), androstenedione (A2) and E2, the activity and gene expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-HSD and the expression of P450 aromatase (Cyp19a1) and (2) whether any such modifications are related to changes in ovarian nitric oxide (NO) and noradrenaline (NA) levels during dioestrus II. Using an ex vivo SMG-ovarian nervous plexus-ovary system, ovarian P4 release was measured following the addition E2 plus tamoxifen (Txf) (10-6M) to the ganglion, whereas A2, E2, NA and NO were measured following the addition of E2 alone. Steroids were measured by radioimmunoassay, NA concentrations were determined by HPLC and gene expression was evaluated using reverse transcription-polymerase chain reaction. Oestradiol in the ganglion decreased ovarian P4, E2 and NA release, as well as 3ß-HSD activity, but increased the release of A2 and nitrites, as well as the 20α-HSD expression and its activity. No changes were observed in Cyp19a1 gene expression. The addition of E2 plus Txf to the ganglion reversed the effects of E2 alone. The action of oestradiol in SMG favours the beginning of functional luteolysis, due to an increase in NO release and a decrease in NA in the ovary. These results may help elucidate the role of E2 in hormone-dependent pathologies in women.

Oxidative stress and altered steroidogenesis in the ovary by cholinergic stimulation of coeliac ganglion in the first proestrous in rats. Implication of nitric oxide.

An ex-vivo Coeliac Ganglion-Superior Ovarian Nerve-Ovary (CG-SON-O) system from virgin rats in the first proestrous was used to test whether cholinergic stimulation of CG affects oxidative status and steroidogenesis in the ovary. The CG and the O were placed in separate buffered-compartments, connected by the SON, and the CG was stimulated by acetylcholine (Ach). To test a possible role of nitric oxide (NO) in the ovarian response to cholinergic stimulation of CG, aminoguanidine (AG) - an inhibitor of inducible-NO synthase was added to the O compartment. After 180 min incubation, the oxidative status was assessed in O whereas nitrite and steroidogenesis were assessed at 30, 120 and 180 min. Ach in CG decreased the total antioxidant capacity, but increased NO production and protein carbonization in O. Ach stimulation of CG increased estradiol, but decreased progesterone release in O by reducing the mRNAs related to their synthesis and degradation. The addition of AG to the O compartment caused an opposite effect, which was more pronounced in the presence of Ach in the CG compartment than in its absence. These results show that the stimulation of the extrinsic-cholinergic innervation of the O increases the concentration of NO, causes oxidative stress and modulates steroidogenesis in the first rat proestrous.

Prolactin modulates luteal regression from the coeliac ganglion via the superior ovarian nerve in the late-pregnant rat.

There is considerable evidence of the neuroendocrine control involved in luteal regression in the rat. In addition, circulating prolactin (PRL), which increases during the night before parturition, may gain access to the coeliac ganglion (CG), indirectly impacting the physiology of the ovary because of the known connection between the CG and the ovary via the superior ovarian nerve (SON). In this work we investigated in the CG-SON-ovary system and whether PRL added to the CG has an impact, indirectly via the SON, on luteal regression on Day 21 of pregnancy. The system was incubated without (control) or with PRL added to the CG. We measured the ovarian release of progesterone (P), oestradiol and prostaglandin F2 alpha (PGF2α) by radioimmunoassay, and nitrites (NO) by the Griess method. Luteal mRNA expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 20α-HSD, aromatase, inducible nitric oxide synthase (iNOS) and apoptosis regulatory factors was analysed by reverse transcription-polymerase chain reaction. P release, the expression of Bcl-2 and the Bcl-2:Bax ratio was lower than control preparations, while the expression of 20α-HSD and the release of NO and PGF2α were higher in the experimental group. In conclusion, PRL acts at the CG and, by a neural pathway, modulates luteal function at the end of pregnancy.

Participation of the extrinsic cholinergic innervation in the action of nitric oxide on the ovarian steroidogenesis in the first proestrous in rats.

An ex-vivo Coeliac Ganglion-Superior Ovarian Nerve-Ovary (CG-SON-O) system and an ovary without peripheral neural influence from virgin rats in the first proestrous were used to test whether ovarian extrinsic innervation and nitric oxide (NO) affects steroidogenesis in the ovary. The CG and the ovary were placed in separate buffered-compartments, connected by the SON. Stimulation of the CG was achieved by 10(-6)M acetylcholine (Ach). The ovary without peripheral neural influence was placed alone in a buffered-compartment. To test a possible role of NO in the ovarian response to peripheral neural influence, 100µM sodium nitroprusside (SNP, an NO donor) and 100µM N(G)-nitro-l-arginine methyl ester (l-NAME, an inhibitor of NO synthase) were added to the ovarian compartment separately. In the CG-SON-O system, SNP into the ovarian compartment increased the concentration of NO, reduced the release of progesterone and increased the release of estradiol (E2), increasing the mRNAs related to their synthesis enzyme. The addition of l-NAME to the ovarian compartment caused an opposite effect. In the ovary alone, NO manifested an antisteroidogenic effect on both hormones. These results show that the ovarian extrinsic innervation maintains a direct relationship between NO and E2, both needed at high levels during the follicular phase, allowing the continuity of the estrous cycle.

Neuromodulation of the luteal regression: presence of progesterone receptors in coeliac ganglion.

NEW FINDINGS: What is the central question of this study? The processes involved in luteal involution have not yet been clarified and, in general, have been studied only from a hormonal point of view. We investigated whether progesterone, from the coeliac ganglion through the superior ovarian nerve, is able to modify the luteal regression of late pregnancy in the rat. What is the main finding and its importance? We showed that the luteal regression might be reversed by the neural effect of progesterone and demonstrated the presence of its receptors in the coeliac ganglion. This suggests that the peripheral neural pathway, through neuron-hormone interaction, represents an additional mechanism to control luteal function in addition to the classical endocrine regulation. The corpus luteum (CL) is a transitory endocrine gland that produces progesterone (P). At the end of its useful life, it suffers a process of functional and structural regression until its complete disappearance from the ovary. To investigate whether P is able to regulate the process of luteal regression through the peripheral neural pathway, we used the coeliac ganglion (CG)-superior ovarian nerve-ovary system from rats on day 21 of pregnancy. We stimulated the CG with P and analysed the functional regression through ovarian P release measured by radioimmunoassay, expression by RT-PCR and activity of luteal 3ß- and 20α-hydroxysteroid dehydrogenase (anabolic and catabolic P enzymes, respectively). The luteal structural regression was evaluated through a study of apoptosis measured by TUNEL assay and the expression of apoptotic factors, such as Bcl-2, Bax, Fas and Fas ligand (FasL) by RT-PCR. To explore whether the effects mediated by P on the CL may be associated with P receptors, their presence in the CG was investigated by immunohistochemistry. In the group stimulated with P in the CG, the ovarian P release and the 3ß-hydroxysteroid dehydrogenase activity increased, whereas the expression and activity of 20α-hydroxysteroid dehydrogenase decreased. In addition, a decrease in the number of apoptotic nuclei and a decrease of the expression of FasL were observed. We demonstrated the presence of P receptors in the CG. Overall, our results suggest that the regression of the CL of late pregnancy may be reprogrammed through the peripheral neural pathway, and this effect might be mediated by P bound to its receptor in the CG.

Estetrol Inhibits Endometriosis Development in an In Vivo Murine Model.

Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus, and it is associated with alterations in the expression of hormone receptors and inflammation. Estetrol (E4) is a weak estrogen that recently has been approved for contraception. We evaluated the effect of E4 on the growth of endometriotic-like lesions and the expression of TNF-α, estrogen receptors (ERs), and progesterone receptors (PRs) in an in vivo murine model. Endometriosis was induced surgically in female C57BL/6 mice. E4 was delivered via Alzet pump (3 mg/kg/day) from the 15th postoperative day for 4 weeks. E4 significantly reduced the volume (p < 0.001) and weight (p < 0.05) of ectopic lesions. Histologically, E4 did not affect cell proliferation (PCNA immunohistochemistry) but it did increase cell apoptosis (TUNEL assay) (p < 0.05). Furthermore, it modulated oxidative stress (SOD, CAT, and GPX activity, p < 0.05) and increased lipid peroxidation (TBARS/MDA, p < 0.01). Molecular analysis showed mRNA (RT-qPCR) and protein (ELISA) expression of TNF-α decreased (p < 0.05) and mRNA expression of Esr2 reduced (p < 0.05), in contrast with the increased expression of Esr1 (p < 0.01) and Pgr (p < 0.05). The present study demonstrates for the first time that E4 limited the development and progression of endometriosis in vivo.

Effect of prolactin acting on the coeliac ganglion via the superior ovarian nerve on ovarian function in the postpartum lactating and non-lactating rat.

Whether prolactin (PRL) has a luteotrophic or luteolytic effect in the rat ovary depends on the nature of the corpora lutea present in the ovaries and the hormonal environment to which they are exposed. The aim was to investigate the effect of PRL acting on the coeliac ganglion (CG) on the function of the corpora lutea on day 4 postpartum under either lactating or non-lactating conditions, using the CG-superior ovarian nerve-ovary system. The ovarian release of progesterone (P), estradiol, PGF2α, and nitrites was assessed in the ovarian compartment at different incubation times. Luteal mRNA expression of 3ß-HSD, 20α-HSD, aromatase, PGF2α receptor, iNOS, Bcl-2, Bax, Fas and FasL was analysed in the corpus luteum of pregnancy at the end of the experiments. Comparative analysis of control groups showed that the ovarian release of P, nitrites, and PGF2α, the expression of PGF2α receptor, and the Bcl-2/Bax ratio were lower in non-lactating rats, with increased release of estradiol, and higher expression of aromatase, Fas and FasL, demonstrating the higher luteal functionality in ovaries of lactating animals. PRL added to the CG compartment increased the ovarian release of P, estradiol, nitrites and PGF2α, and decreased the Bcl-2/Bax ratio in non-lactating rats; yet, with the exception of a reduction in the release of nitrites, such parameters were not modified in lactating animals. Together, these data suggest that the CG is able to respond to the effect of PRL and, via a neural pathway, fine-tune the physiology of the ovary under different hormonal conditions.

The copper chelator ammonium tetrathiomolybdate inhibits the progression of experimental endometriosis in TNFR1-deficient mice.

The TNF-α/TNFR system is involved in endometriosis (EDT), a gynecologic estrogen-dependent disease. Elevated copper concentrations have also been associated with EDT, even in TNFR1-deficient mice where disease worsening occurs. We aimed to evaluate whether treatment with ammonium tetrathiomolybdate (TM, copper chelator) is beneficial in TNFR1-deficient mice presenting with worsened EDT status. Female C57BL/6 mice were divided into three groups: KO Sham, KO EDT, and KO EDT+TM. TM was administered from the 15th postoperative day, and samples were collected one month after inducing pathology. In peritoneal fluid, copper and estradiol levels were determined by electrothermal atomic absorption spectrometry and electrochemiluminescence, respectively. Lesions were processed for the analysis of cell proliferation (PCNA immunohistochemistry), expression of angiogenic markers (RT-qPCR), and oxidative stress (spectrophotometric methods). We found that EDT increased copper and estradiol levels compared to the KO Sham group, while the TM administration restored the levels of both factors. TM also reduced the volume and weight of the lesions and cell proliferation rate. Besides, TM treatment decreased the number of blood vessels and the Vegfa, Fgf2, and Pdgfb expression. Furthermore, superoxide dismutase and catalase activity decreased, and lipid peroxidation increased. TM administration inhibits EDT progression in TNFR1-deficient mice where the pathology is exacerbated.

Superior mesenteric ganglion via ovarian plexus nerve involved in the cross-talk between noradrenaline and GnRH in rat ovaries.

There is evidence of the existence of an intraovarian gonadotropin-releasing hormone (GnRH) system. There are also reports about the influence of extrinsic ovarian innervation in gonadal function. Therefore, it is interesting to study the relationship between ovarian sympathetic innervation and GnRH to shed light on possible physiological and pathophysiological implications. This work aimed to investigate whether noradrenergic stimulation of the superior mesenteric ganglion (SMG) can modify the levels of ovarian GnRH and cause functional and morphological changes in the gonad through the ovarian plexus nerve (OPN), during estrus and diestrus II in rats. The SMG-OPN-Ovary system and an ovary without extrinsic innervation were removed from Holtzman rats in estrus and diestrus II stages and placed in specially designed cuvettes containing Krebs-Ringer buffer. In the experimental groups, SMGs and denervated ovaries were stimulated with 10-6 M noradrenaline (NA). GnRH and progesterone levels (in the ovarian incubation medium) and the mRNA expression of 3beta-hydroxysteroid dehydrogenase (Hsd3b3), 20alpha-hydroxysteroid dehydrogenase (Akr1c18), Bax, and Bcl2 were analyzed. Histological studies of the ovaries were performed. In estrus, NA decreased GnRH levels in both experimental schemes. Furthermore, progesterone levels increased while the Akr1c18 expression and Bax/Bcl2 ratio decreased, without causing changes in ovarian morphology. In diestrus, the noradrenergic stimulation of the ganglion increased GnRH levels, decreased progesterone levels, and increased Akr1c18 expression and Bax/Bcl2 ratio. Follicles with histoarchitecture alterations and corpus luteum with signs of cell death were observed. In denervated ovaries, NA increased the levels of GnRH and progesterone. Furthermore, NA decreased the Bax/Bcl2 ratio and histological studies revealed signs compatible with a possible atretogenic effect. In conclusion, noradrenergic stimulation of the SMG-OPN pathway regulates ovarian cyclicity. The SMG modulates the cross-talk between NA and ovarian GnRH, protecting the ovary from atretogenic effects and luteal apoptosis during estrus while inducing luteal regression in the diestrus II.

Effect of peripheral neural stimulation with allopregnanolone on ovarian physiology.

Neuroactive steroids can rapidly regulate multiple physiological functions in the central and peripheral nervous systems. The aims of the present study were to determine whether allopregnanolone (ALLO), administered in low nanomolar and high micromolar concentrations, can: (i) induce changes in the ovarian progesterone (P4) and estradiol (E2) release; (ii) modify the ovarian mRNA expression of Hsd3b1 (3ß-hydroxysteroid dehydrogenase, 3ß-HSD)3ß-, Akr1c3 (20α-hydroxysteroid dehydrogenase, 20α-HSD), and Akr1c14 (3α-hydroxy steroid oxidoreductase, 3α-HSOR)); and (iii) modulate the ovarian expression of progesterone receptors A and B, α and ß estrogenic receptors, luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). To further characterize ALLO peripheral actions, the effects were evaluated using a superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) and a denervated ovary (DO) systems. ALLO SMG administration increased P4 concentration in the incubation liquid by decreasing ovarian 20α-HSD mRNA, and it also increased ovarian 3α-HSOR mRNA expression. In addition, ALLO neural peripheral modulation induced an increase in the expression of ovarian LHR, PRA, PRB, and ERα. Direct ALLO administration to the DO decreased E2 and increased P4 concentration in the incubation liquid. The mRNA expression of 3ß-HSD decreased and 20α-HSD increased. Further, ALLO in the OD significantly changed ovarian FSHR and PRA expression. This is the first evidence of ALLO's direct effect on ovarian steroidogenesis. Our results provide important insights about how this neuroactive steroid interacts both with the PNS and the ovary, and these findings might help devise some of the pleiotropic effects of neuroactive steroids on female reproduction. Moreover, ALLO modulation of ovarian physiology might help uncover novel treatment approaches for reproductive diseases.

Superior mesenteric ganglion neural modulation of ovarian angiogenesis, apoptosis and proliferation by the neuroactive steroid allopregnanolone.

Allopregnanolone (ALLO), a potent neuroactive steroid, is synthesized and active in the peripheral nervous system. Previous studies have shown that ALLO participates in the central regulation of reproduction with effects on ovarian physiology, although there is little evidence for its ability to modulate peripheral tissues. The present study aimed to determine whether ALLO, administered to an ex vivo system that comprises the superior mesenteric ganglion (SMG), the ovarian nervous plexus (ONP) and the ovary (O), or to the denervated ovary (DO), was able to modify ovarian apoptosis, proliferation and angiogenesis. For this purpose, the SMG-ONP-O system and DO were incubated during 120 min at 37°C, in the presence of two ALLO doses (0.06 µm and 6 µm). The intrinsic and extrinsic pathways of apoptosis were analyzed. Incubation of the SMG-ONP-O system with ALLO 0.06 µm led to an increase in the BAX/BCL-2 ratio and a reduction of FAS-L mRNA levels. ALLO 6 µm induced a decrease of FAS-L levels. Incubation of DO with ALLO 0.06 µm reduced FAS-L, whereas ALLO 6 µm significantly increased it. Cyclin D1 mRNA was measured to evaluate proliferation. Treatment with ALLO 6 µm increased proliferation in both SMG-ONP-O and DO. ALLO 0.06 µm produced an increase of Cyclin D1 in DO only. Administration of either ALLO dose led to a higher ovarian expression of vascular endothelial growth factor in the SMG-ONP-O system, but a lower one in the DO system. ALLO 6 µm induced ovarian sensitization to GABA by increasing GABAA receptor expression. In conclusion, ALLO participates in the peripheral neural modulation of ovarian physiology. It can also interact directly with the ovarian tissue, modulating key mechanisms involved in normal and pathological processes in a dose-dependent manner.

Antiproliferative and antiangiogenic effects of ammonium tetrathiomolybdate in a model of endometriosis.

AIMS: Copper (Cu) is involved in the endometriosis progression. Herein, an experimental endometriosis model was used to evaluate whether its chelation with ammonium tetrathiomolybdate (TM) affects the proliferation and angiogenesis in endometriotic-like lesions and the participation of oxidative stress in these processes. MAIN METHODS: Female C57BL/6 mice were divided into three groups: sham-operated mice, endometriosis-induced mice, and TM-treated endometriosis-induced mice. Each animal in the third group received 0.3 mg of TM/day in their drinking water from the postoperative 15th day. The samples were collected after one month of induced pathology. In peritoneal fluids, Cu and estradiol levels were determined by electrothermal atomic absorption spectrometry and electrochemiluminescence, respectively. Endometriotic-like lesions were processed for the analysis of cell proliferation by PCNA immunohistochemistry, the expression of angiogenic markers by RT-qPCR, the presence of endothelial cells by immunofluorescent staining, and oxidative stress applying spectrophotometric methods. KEY FINDINGS: TM treatment decreased Cu and estradiol levels, which were increased by this pathology. In lesions, TM induced: (a) a decrease in tissue weight and volume, (b) a decrease in PCNA-positive cells, (c) antiangiogenic effects by decreasing the number of blood vessels, the mRNA expression of fibroblast growth factor 2 (Fgf2) and platelet-derived growth factor subunit B (Pdgfb), and the presence of endothelial cells, (d) a decrease in antioxidant activity and an increase in lipid peroxidation. SIGNIFICANCE: TM is a highly effective antiproliferative and antiangiogenic agent, modulating oxidative imbalance in endometriosis. Its anti-endometriotic potential is an attractive feature of TM as a possible non-hormonal treatment.

Effect of an Intracerebroventricular Injection of Aggregated Beta-amyloid (1-42) on Daily Rhythms of Oxidative Stress Parameters in the Prefrontal Cortex.

Accumulation of amyloid peptides in the brain plays a key role in the pathogenesis of Alzheimer's disease (AD). Aggregated beta-amyloid (Aß) peptide increases intracellular reactive oxygen species associated to a deficient antioxidant defense system. Prefrontal cortex plays a key role in memory and learning and is especially susceptible to oxidative stress. The objective of this work was to investigate the effects of an intracerebroventricular (i.c.v.) injection of Aß (1-42) on 24 h patterns of oxidative stress parameters and antioxidant defenses in the rat prefrontal cortex. Four-month-old male Holtzman rats were divided into two groups defined as: control (CO) and Aß-injected (Aß). Rats were maintained under12 h-light:12 h-dark conditions and received water and food ad libitum. Tissues samples were isolated every 6 h during a 24 h period. Interestingly, we found that an i.c.v. injection of Aß(1-42) increased lipid peroxidation, reduced total antioxidant capacity level, phase-shifted the daily peak of reduced glutathione, and had a differential effect on the oscillating catalase and glutathione peroxidase specific activity. Thus, elevated levels of Aß aggregates-a pathogenic hallmark of AD, caused altered temporal patterns of the cellular redox state in prefrontal cortex rat. These findings might contribute, at least in part, to the understanding of the molecular and biochemical basis of redox changes caused by circadian rhythms alterations observed in AD patients.

An intracerebroventricular injection of amyloid-beta peptide (1-42) aggregates modifies daily temporal organization of clock factors expression, protein carbonyls and antioxidant enzymes in the rat hippocampus.

Alzheimer disease (AD) is the most frequent form of dementia in the elderly. It is characterized by the deterioration of memory and learning. The histopathological hallmarks of AD include the presence of extracellular deposits of amyloid beta peptide, intracellular neurofibrillary tangles, neuron and synapse loss, in the brain, including the hippocampus. Accumulation of Aß peptide causes an increase in intracellular reactive oxygen species (ROS) and free radicals associated to a deficient antioxidant defense system. Besides oxidative stress and cognitive deficit, AD patients show alterations in their circadian rhythms. The objective of this work was to investigate the effects of an intracerebroventricular injection of amyloid beta peptide Aß(1-42) aggregates on temporal patterns of protein oxidation, antioxidant enzymes and clock factors in the rat hippocampus. Four-month-old male Holtzman rats divided into the groups control (CO) and Aß-injected (Aß), were maintained under 12 h-light12h-dark conditions and received water and food ad-libitum. Hippocampus samples were isolated every 6 h during a 24 h period. Our results showed daily patterns of protein carbonyls, catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as Rorα and Rev-erbß mRNA, in the rat hippocampus. Interestingly, an intracerebroventricular injection of Aß aggregates modified daily oscillation of protein carbonyls levels, phase-shifted daily rhythms of clock genes and had a differential effect on the daily expression and activity of CAT and GPx. Thus, Aß aggregates might affect clock-mediated transcriptional regulation of antioxidant enzymes, by affecting the formation of BMAL1:CLOCK heterodimer, probably, as a consequence of the alteration of the redox state observed in rats injected with Aß.

Androgen receptors in coeliac ganglion in late pregnant rat.

The ovarian function is controlled by endocrine factors and neural influence. In late pregnant rat, androstenedione, from the coeliac ganglion, has a luteotrophic effect in the ex vivo coeliac ganglion-superior ovarian nerve-ovary system. In this work we investigate the presence of androgen receptors in the coeliac ganglion of late pregnant rats by immunohistochemistry. We also explore, from a physiological point of view, the potential participation of these receptors in the androstenedione ganglionic action on progesterone release and metabolism, as well as on nitrites release in the ovary compartment. The coeliac ganglion was isolated after being fixed in situ and immunohistochemistry was performed. In the system, three experimental groups were used with the addition of (a) androstenedione, (b) flutamide, and (c) androstenedione plus flutamide in the ganglion compartment. Progesterone and nitrite concentrations were determined in the ovary compartment at different incubation times. Corpora lutea samples isolated at the end of incubation were used to determine the expressions and activities of the progesterone synthesis (3beta-hydroxysteroid-dehydrogenase, 3beta-HSD) and degradation (20alpha-hydroxysteroid-dehydrogenase, 20alpha-HSD) enzymes. Immunohistochemistry revealed cytoplasmatic androgen receptor immunoreactivity in neural somas in the coeliac ganglion. In the coeliac ganglion-superior ovarian nerve-ovary system, androstenedione addition increased 3beta-HSD and decreased 20alpha-HSD, showed a tendency to decrease 20alpha-HSD expression, and increased nitrites release in relation to control. Androstenedione plus flutamide decreased progesterone and nitrites release in relation to the androstenedione group. This work demonstrates the presence of androgen receptors in neurons of celiac ganglion and provides evidence for the luteotrophic action of androstenedione via a neural pathway that may be mediated by these receptors.