Acute restraint stress regulates brain DNMT3a and promotes defensive behaviors in male rats.

Depending on its duration and severity, stress may contribute to neuropsychiatric diseases such as depression and anxiety. Studies have shown that stress impacts the hypothalamic-pituitary-adrenal (HPA) axis, but its downstream molecular, behavioral, and nociceptive effects remain unclear. We hypothesized that a 2-hour single exposure to acute restraint stress (ARS) activates the HPA axis and changes DNA methylation, a molecular mechanism involved in the machinery of stress regulation. We further hypothesized that ARS induces anxiety-like and risk assessment behavior and alters nociceptive responses in the rat. We employed biochemical (radioimmunoassay for corticosterone; global DNA methylation by enzyme immunoassay and western blot for DNMT3a expression in the amygdala, ventral hippocampus, and prefrontal cortex) and behavioral (elevated plus maze and dark-light box for anxiety and hot plate test for nociception) tests in adult male Wistar rats exposed to ARS or handling (control). All analyses were performed 24 h after ARS or handling. We found that ARS increased corticosterone levels in the blood, increased the expression of DNMT3a in the prefrontal cortex, promoted anxiety-like and risk assessment behaviors in the elevated plus maze, and increased the nociceptive threshold observed in the hot plate test. Our findings suggest that ARS might be a helpful rat model for studying acute stress and its effects on physiology, epigenetic machinery, and behavior.

Imipramine attenuates anxiety- and depressive-like effects of acute and prolonged ethanol-abstinence in male rats by modulating SERT and GR expression in the dorsal hippocampus.

AIMS: Considering that serotoninergic agents attenuate symptoms of anxiety and are used to treat depression, we investigated whether subchronic treatment with imipramine, a serotonin/noradrenaline reuptake inhibitor, would prevent the anxiogenic-like behaviour induced by acute and/or chronic ethanol withdrawal. We also investigated whether those changes were related to the disfunctioning of hypothalamic-pituitary-adrenal (HPA) axis and serotonergic neurotransmission. MAIN METHODS: 264 Male Wistar rats were treated with ethanol 6% (vol./vol.) for 21 days. Acute ethanol withdrawal was induced by abrupt discontinuation of treatment and sustained for 48 h. Protracted abstinence was sustained for an additional period of 21 days. Behavioural tests included the Elevated Plus Maze (EPM) or Light/Dark Box (LDB) after acute abstinence, and the Forced Swim Test (FST) after protracted abstinence. Imipramine (15 mg/kg, i.p.) was administered 24, 19 and 1 h before EPM or LDB tests. KEY FINDINGS: Acute abstinence decreased exploration of the open arms of the EPM, without changing exploration of LDB. Additionally, chronic abstinent rats displayed more time immobile in the FST, when compared to control animals. These effects were attenuated by imipramine treatment, without changing basal response. Imipramine prevented protracted abstinence -induced decrease in glucocorticoid receptor (GR) and serotonin transporter (SERT) expression in the dorsal hippocampus. SIGNIFICANCE: Our findings indicate that chronic ethanol withdrawal affects the hippocampal serotonergic system by decreasing serotonin transporter expression. It also disturbs the HPA axis functioning through an imbalance on GR and mineralocorticoid (MR) expression.

Impact of timing of the anorexigen sibutramine administration on reproductive end-points of male rats.

Sibutramine is a non-selective serotonin-norepinephrine reuptake inhibitor orally administered for weight loss. In a previous study, we showed pharmacological mechanisms involved in the reduction of sperm quality and fertility of rats exposed for 30 days to this anorexigen in the light phase of the light-dark (l/d) cycle. It is already known that rodents are nightlife animals, with higher metabolic activity during the dark phase than in the light phase of the light-dark (l/d) cycle. Thus, the present study aimed to investigate whether the deleterious effects on reproductive parameters after sibutramine administration would be enhanced after a shorter period of exposure during the dark phase of the l/d cycle. For this, adult male Wistar rats were treated with sibutramine (10 mg/kg/d) or vehicle for 15 days during the dark phase of the l/d cycle. Sibutramine treatment decreased final body and reproductive organ weights, as well as serum testosterone levels. Sperm transit time through the epididymis was accelerated, and sperm concentration and motility were diminished in the sibutramine-exposed rats. The decrease in sperm concentration was also verified in the epididymal histological sections. In conclusion, the deleterious effects of sibutramine on reproductive parameters of male rats were enhanced when the exposure occurred in the dark phase of the l/d cycle, even after a short exposure duration. Our results reinforce the impact of timing on drug therapeutic action.

Short- and long-term effects on reproductive parameters of female Wistar rats after exposure to rosuvastatin starting in pre-puberty.

Statins are a class of drugs that act lowering lipid levels by inhibiting cholesterol biosynthesis. Additionally, statins can act by "pleiotropic effects", related to the inhibition of synthesis of the other mevalonate pathway products. Rosuvastatin is a third-generation statin and has shown better results in reducing cholesterol concentrations when compared to other statins. Recent studies suggest that rosuvastatin may act as an endocrine disruptor that potentially damages the hormonal axis and, consequently reproductive development and function of male rats. However, the effects of rosuvastatin exposure on rat female reproductive parameters remain unknown. In this study female rats were exposed to rosuvastatin at the doses of 0 (control), 3, or 10 mg/Kg.bw-1/day from pre-puberty to adulthood. No alterations in the female reproductive parameters were observed at a dose of 3 mg/Kg.bw-1. However, females exposed to 10 mg/Kg.bw-1 exhibited shorter estrous cycles, altered copulatory behavior, decreased serum prolactin level, and alterations in the liver, pituitary and placental weights, parameters to some extent influenced by the reproductive hormonal axis signaling pathway. On the other hand, pubertal onset, reproductive hormone levels, fertility, and histological parameters of the ovary, uterus, and placenta were unaltered by exposure to both doses of this statin. Thus, rosuvastatin exposure, at the higher dose, altered the reproductive function of female rats, probably due to the pleiotropic effects of this statin. Additional studies on the effects of this statin on female reproductive function and development are encouraged to better characterize its mode of action.

Citral-induced analgesia is associated with increased spinal serotonin, reduced spinal nociceptive signaling, and reduced systemic oxidative stress in arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Citral (3,7-dimethyl-2,6-octadienal) is the main component of Cymbopogon citratus (DC) Stapf, an herb with analgesic properties. Arthritic pain is the main unpleasant component of rheumatoid arthritis. The pharmacological approaches used to treat arthritic pain are often accompanied by adjuvant drugs or non-pharmacological treatments, showing a constant need in identifying new efficient analgesic drugs. AIM OF THE STUDY: To test the hypothesis that citral, which is a monoterpenoid compound with therapeutic properties, reduces nociception, spinal pro-nociceptive and pro-inflammatory signaling, and systemic oxidative stress in arthritic rats. MATERIALS AND METHODS: Complete Freund's adjuvant (CFA) was administrated in the left knee joint of rats. Oral treatment with citral was performed during eight days and mechanical allodynia was monitored during the period of treatment to evaluate the analgesic effect of citral. We assessed the levels of serotonin (5-hydroxytryptamine, 5-HT) in the lumbar dorsal horn of the spinal cord (DHSC) and the profiles of expression of the glycogen synthase kinase-3ß (GSK3ß), which is a 5-HT-regulated intracellular protein, and of the stress-activated protein kinase (SAPK)/jun N-terminal kinase (JNK) in the DHSC. Plasma levels of superoxide dismutase (SOD) were assessed as an indicator of oxidative stress. RESULTS: Administration of CFA induced mechanical allodynia associated with reduced spinal GSK3ß phosphorylation, increased spinal SAPK/JNK phosphorylation, and increased plasma SOD levels. Oral administration of citral reversed mechanical allodynia, increased endogenous spinal 5-HT levels, reduced spinal SAPK/JNK phosphorylation, and reduced plasma SOD levels. CONCLUSION: Citral shows anti-nociceptive effects in an animal model of arthritic pain by modulating spinal nociceptive signaling.

Ovarian failure induced by 4-vinylcyclohexene diepoxide worsens the autonomic cardiovascular response to chronic unpredictable stress in rats.

AIMS: After menopause, women are more responsive to stress and more prone to exhibit hypertension, which elevates the risk of cardiac diseases. This vulnerability is due, in part, to the decline of ovarian steroids plasma levels. The 4-vinylciclohexane diepoxide (VCD) causes a gradual depletion of ovarian follicles causing loss of the normal ovarian function and a hormonal profile comparable to menopause in humans. We aimed to verify whether the ovarian failure (OF) worsens the cardiovascular autonomic response to stress. MAIN METHODS: Rats were treated with VCD (160 mg/kg) or oil for 15 days, exposed to chronic unpredictable stress (CUS) for 10 days and studied 80 and 180 days after VCD treatment. KEY FINDINGS: 80 days after VCD-treatment, stressed rats showed increased sympathetic nerve activity, reduced parasympathetic activity and an increase in the overall spontaneous baroreflex sensitivity (BRS). 180 days after VCD treatment, BRS was impaired and the vascular sympathetic activity was increased, independently of stress exposure. SIGNIFICANCE: Neither 80 nor 180 days after the onset of VCD-treatment the hypertensive effects of stress were enhanced in rats. However, OF led to a worsening on different aspects of the cardiovascular response to stress, which can cause cardiovascular complications when associated with ovarian aging.

Central serotonin prevents hypotension and hypothermia and reduces plasma and spleen cytokine levels during systemic inflammation.

An exceptionally high mortality rate is observed in sepsis and septic shock. Systemic administration of lipopolysaccharide (LPS) has been used as an experimental model for sepsis resulting in an exacerbated immune response, brain neurochemistry adjustments, hypotension, and hypothermia followed by fever. Central serotonergic pathways not only modulate systemic inflammation (SI) but also are affected by SI, including in the anteroventral region of the hypothalamus (AVPO), which is the hierarchically most important region for body temperature (Tb) control. In this study, we sought to determine if central serotonin (5-HT) plays a role in SI induced by intravenous administration of LPS (1.5 mg/kg) in male Wistar rats (280-350 g) by assessing 5-HT levels in the AVPO, mean arterial pressure, heart rate, and Tb up to 300 min after LPS administration, as well as assessing plasma and spleen cytokine levels, nitric oxide (NO) plasma levels, and prostaglandin (PG) E2 levels in the AVPO at 75 min and 300 min after LPS administration. We observed reduced AVPO 5-HT levels, hypotension, tachycardia, hypothermia followed by fever, as well as observing increased plasma NO, plasma and spleen cytokines and AVPO PGE2 levels in SI. Intracerebroventricular (icv) administration of 5-HT 30 min before LPS administration prevented hypotension and hypothermia, which were accompanied by reduced plasma NO, as well as plasma TNF-α, IL-1ß, IL-6, and IL-10 and spleen TNF-α and IL-10 levels. We suggest that SI reduced 5-HT levels in the AVPO favor an increased pro-inflammatory status both centrally and peripherally that converge to hypotension and hypothermia. Moreover, our results are consistent with the notion that exogenous 5-HT given icv prevents hypotension and hypothermia probably activating the splenic anti-inflammatory pathway.

Sleep restriction during peripuberty unbalances sexual hormones and testicular cytokines in rats.

Spermatogenesis and steroidogenesis are not fully established during puberty. Especially during this period, children and adolescents may be chronically sleep deprived due to early school hours and constant exposure to artificial light and interactive activities. We have previously shown that sleep restriction (SR) during peripuberty impairs sperm motility and has consequences on epididymal development in rats. Thus, this study aimed to evaluate the effect of SR during peripuberty on sexual hormones and its impact on testicular tissue. Rats were subjected to 18 h of SR per day for 21 days or were maintained as controls (C) in the same room. The circulating luteinizing hormone levels were decreased in SR rats without changes in the follicle stimulating hormone levels. Plasma and intratesticular testosterone and corticosterone in the SR group were increased in relation to C group. These alterations impair testicular tissue, with decreased IL-1ß, IL-6, and TNFα levels in the testis and diminished seminiferous epithelium height and Sertoli cell number. SR also increased testicular lipid peroxidation with no alteration in antioxidant profiles. There were no significant changes in sperm parameters, seminiferous tubule diameter, histopathology, spermatogenesis kinetics, neutrophil and macrophage recruitment, and IL-10 concentration. Our results show that SR unbalances sexual hormones and testicular cytokines at a critical period of sexual maturation. These changes lead to lipid peroxidation in the testes and negatively influence the testicular tissue, as evidenced by diminished seminiferous epithelium height-with apoptosis of germinative cell-and Sertoli cell number.

Protective effect of resveratrol on urogenital sinus and prostate development in rats exposed in utero to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin).

This study evaluated the protective effects of resveratrol on the prostate development of rats exposed to TCDD. Pregnant rats received TCDD (1 µg/kg) at GD15 and/or RES (20 mg/kg/day) from GD10 to PND21. Newborn and adult males from Control, TCDD, TCDD + RES and RES groups were euthanized and the prostate was excised. On PND1, there was a reduction in the number of prostatic buds, AR-positive mesenchymal cells and proliferation index in epithelial and mesenchymal cells in TCDD group, but restored by RES. AhR immunoreactivity was greater in TCDD group than the other groups. On PND90, there was higher frequency of functional hyperplasia in the distal area of the prostate acini in TCDD group, but restored by RES. AhRR expression was higher in the TCDD while NRF2 was higher in the TCDD + RES compared to the other groups. Resveratrol was able to reduce the adverse effects of TCDD on prostate development and its long-term repercussions.

Chronic exposure to cannabidiol induces reproductive toxicity in male Swiss mice.

Children and adults with frequent and severe episodes of epilepsy that do not respond to standard treatments (such as carbamazepine, phenytoin and valproate) have long been prescribed cannabidiol (CBD) as an anticonvulsant drug. However, the safety of its chronic use in relation to reproduction has not been fully examined. This study aimed to assess the effects of chronic CBD exposure on the male reproductive system. CBD was orally administered to 21-day-old male Swiss mice at doses of 15 and 30 mg kg-1 daily (CBD 15 and 30 groups, respectively), with a control group receiving sunflower oil, for 34 consecutive days. After a 35 day recovery period, the following parameters were evaluated: weight of reproductive organs, testosterone concentration, spermatogenesis, histomorphometry, daily sperm production and its morphology. The CBD 30 group had a 76% decrease in total circulating testosterone, but it remained within the physiological normal range (240-1100 ng dl-1 ). CBD treatment induced a significant increase in the frequency of stages I-IV and V-VI of spermatogenesis, and a decrease in the frequency of stages VII-VIII and XII. A significant decrease in the number of Sertoli cells was observed only in the CBD 30 group. In both CBD groups the number of spermatozoa in the epididymis tail was reduced by 38%, sperm had head abnormalities, and cytoplasmic droplets were observed in the medial region of flagellum. These results indicated that chronic CBD exposure was associated with changes in the male reproductive system, suggesting its reproductive toxicity.

Modulation of inflammatory and hormonal parameters in response to testosterone therapy: Effects on the ventral prostate of adult rats.

Testosterone is often recommended in the treatment of several aging-related conditions. However, there are still questions about the consequences of this therapy in terms of hormonal and inflammatory parameters that are crucial for prostate homeostasis. Thus, we investigate if the testosterone therapy (TT) modulates the hormone receptors and inflammatory cytokines in the ventral prostate of adult rats. Wistar rats aging 150 days were divided into two experimental groups (n = 10/group): T: received subcutaneous injections of testosterone cypionate (5 mg/kg body weight) diluted in corn oil every other day for 4 weeks; and C: received corn oil as vehicle. Animals were euthanized at 180 days old by decapitation. Blood was collected to obtain hormone and cytokines concentrations. The ventral prostate was dissected and processed for light microscope and molecular analyses. Relative ventral prostate weight and epithelial compartment were increased after TT. The number of intact and degranulated mast cells was reduced in the T group. Plasma testosterone, DHT and intraprostatic testosterone concentrations were higher in the T group. TT leads to an increase in cell proliferation and up-regulation of AR, ERß, PAR-4, and NRF2. Importantly, plasma concentration and tissue expression of IL-10 and TNF-α were higher after TT. In summary, these results indicate that TT can regulate inflammatory response, with impacts in cytokines and mast cell population, and modulates steroids receptors, important parameters for prostatic homeostasis.

Effects of Estrogen Therapy on the Serotonergic System in an Animal Model of Perimenopause Induced by 4-Vinylcyclohexen Diepoxide (VCD).

Chronic exposure to 4-vinylcycloxene diepoxide (VCD) in rodents accelerates the natural process of ovarian follicular atresia modelling perimenopause in women. We investigated why estrogen therapy is beneficial for symptomatic women despite normal or high estrogen levels during perimenopause. Female rats (28 d) were injected daily with VCD or oil for 15 d; 55-65 d after the first injection, pellets of 17ß-estradiol or oil were inserted subcutaneously. Around 20 d after, the rats were euthanized (control rats on diestrus and estradiol-treated 21 d after pellets implants). Blood was collected for hormone measurement, the brains were removed and dorsal raphe nucleus (DRN), hippocampus (HPC), and amygdala (AMY) punched out for serotonin (5-HT), estrogen receptor ß (ERß), and progesterone receptor (PR) mRNA level measurements. Another set of rats was perfused for tryptophan hydroxylase (TPH) immunohistochemistry in the DRN. Periestropausal rats exhibited estradiol levels similar to controls and a lower progesterone level, which was restored by estradiol. The DRN of periestropausal rats exhibited lower expression of PR and ERß mRNA and a lower number of TPH cells. Estradiol restored the ERß mRNA levels and number of serotonergic cells in the DRN caudal subregion. The 5-HT levels were lower in the AMY and HPC in peristropausal rats, and estradiol treatment increased the 5-HT levels in the HPC and also increased ERß expression in this area. In conclusion, estradiol may improve perimenopause symptoms by increasing progesterone and boosting serotonin pathway from the caudal DRN to the dorsal HPC potentially through an increment in ERß expression in the DRN.

Cardiovascular Autonomic Responses in the VCD Rat Model of Menopause: Effects of Short- and Long-Term Ovarian Failure.

After menopause, hypertension elevates the risk of cardiac diseases, one of the major causes of women's morbidity. The gradual depletion of ovarian follicles in rats, induced by 4-vinylcyclohexene diepoxide (VCD), is a model for studying the physiology of menopause. 4-Vinylcyclohexene diepoxide treatment leads to early ovarian failure (OF) and a hormonal profile comparable to menopause in humans. We have hypothesized that OF can compromise the balance between sympathetic and parasympathetic tones of the cardiovascular system, shifting toward dominance of the former. We aimed to study the autonomic modulation of heart and blood vessels and the cardiovascular reflexes in rats presenting short-term (80 days) or long-term (180 days) OF induced by VCD. Twenty-eight-day-old Wistar rats were submitted to VCD treatment (160 mg/kg, intraperitoneally) or vehicle (control) for 15 consecutive days and experiments were conducted at 80 or 180 days after the onset of treatment. Long-term OF led to an increase in the sympathetic activity to blood vessels and an impairment in the baroreflex control of the heart, evoked by physiological changes in arterial pressure. Despite that, long-term OF did not cause hypertension during the 180 days of exposure. Short-term OF did not cause any deleterious effect on the cardiovascular parameters analyzed. These data indicate that long-term OF does not disrupt the maintenance of arterial pressure homeostasis in rats but worsens the autonomic cardiovascular control. In turn, this can lead to cardiovascular complications, especially when associated with the aging process seen during human menopause.

Participation of locus coeruleus in breathing control in female rats.

Several evidences indicate that the locus coeruleus (LC) is involved in central chemoreception responding to CO2/pH and displaying a high percentage of chemosensitive neurons (>80%). However, there are no studies about the LC-mediated hypercapnic ventilation performed in females. Therefore, we assessed the role of noradrenergic LC neurons in non-ovariectomized (NOVX), ovariectomized (OVX) and estradiol (E2)-treated ovariectomized (OVX+E2) rats in respiratory response to hypercapnia, using a 6-hydroxydopamine (6-OHDA) - lesion model. A reduction in the number of tyrosine hydroxylase (TH) immunoreactive neurons (51-90% in 3 animals of NOVX group, 20-42% of lesion in 5 animals of NOVX females, 61.3% for OVX and 62.6% for OVX+E2 group) was observed seven days after microinjection of 6-OHDA in the LC. The chemical lesion of the LC resulted in decreased respiratory frequency under normocapnic conditions in OVX and OVX+E2 group. Hypercapnia increased ventilation in all groups as consequence of increases in respiratory frequency (fR) and tidal volume (VT). Nevertheless, the hypercapnic ventilatory response was significantly decreased in 6-OHDA-NOVX>50% rats compared with SHAM-NOVX group and with females that had 20-42% of LC lesion. In OVX and OVX+E2 lesioned groups, no difference in CO2 ventilatory response was observed when compared to SHAM-OVX and SHAM-OVX+E2 groups, respectively. Neither basal body temperature (Tb) nor Tb reduction in response to hypercapnia were affected by E2 treatment, ovariectomy or LC lesion. Thus, our data show that LC noradrenergic neurons seem to exert an excitatory role on the hypercapnic ventilatory response in female rats, as evidenced by the results in NOVX animals with LC lesioned more than 50%; however, this modulation is not observed in OVX and OVX+E2 rats. In addition, LC noradrenergic neurons of OVX females seem to provide a tonic excitatory drive to maintain breathing frequency in normocapnia, and this response may not to be functionally influenced by E2.

Central serotonin attenuates LPS-induced systemic inflammation.

Serotonin (5-HT) is a neuromodulator involved in several central-mediated mechanisms, such as endocrine processes, behavior, and sleep. Dysfunction of the serotonergic system is mainly linked to psychiatric disorders, but emerging evidence suggests that immune system activation may also alter brain 5-HT signaling. However, whether central 5-HT modulates systemic inflammation (SI) remains unknown. For this purpose, male Wistar rats (280-350g, 8-9weeks) were submitted to the experimental protocols beginning between 9 and 10AM with the performance of injections. The animals were housed at controlled conditions [temperature (25±1°C), light (06:00-18:00) and humidity (60-65%)]. Thus, we measured 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the anteroventral preoptic region [(AVPO) - the hierarchically most important region for body temperature (Tb) control] during lipopolysaccharide (LPS)-induced SI. We also combined LPS (100µg/kg) treatment with intracerebroventricular (icv) injection of 5-HT (5, 10 and 40µg/µL) and measured Tb ("hallmark" of SI), AVPO prostaglandin E2 [(PGE2) - an essential mediator of fever] and prostaglandin D2 [(PGD2) - a cryogenic mediator], plasma corticosterone [(CORT) - a stress marker with an endogenous anti-inflammatory effect] and interleukin-6 [(IL-6) - an immune mediator] levels. Detection limits of PGE2, PGD2, CORT and IL-6 assays were 39.1-2500pg/mL, 19.5-2500pg/mL, 0.12-2000µg/dL, and 0.125-8ng/mL, respectively. We also assessed tail skin temperature [used to calculate heat loss index (HLI)] to assess a key thermoeffector mechanism. As expected we observed LPS-induced increases in Tb, AVPO PGE2 (whereas PGD2 remained unchanged), plasma CORT and IL-6 levels, as well as a decrease in HLI. These changes were accompanied by reduced levels of AVPO 5-HT and 5-HIAA. Furthermore, we also observed a negative correlation between 5-HT and plasma CORT levels. Moreover, icv 5-HT (5, 10 and 40µg/µL) microinjection caused a U-shaped dose-response curve in LPS fever, in which the intermediate dose reduced the febrile response. Icv 5-HT (10µg/µL) microinjection prevented the LPS-induced increases in AVPO PGE2 (whereas not altering PGD2), plasma CORT and IL-6 levels, as well as preventing reduced HLI. Our data are consistent with the notion that AVPO 5-HT synthesis is down-regulated during SI, favoring AVPO PGE2 synthesis and consequently potentiating the immune response. These results reveal a novel effect of central 5-HT as an anti-inflammatory neuromodulator that may take place during psychiatric disorder treatment with 5-HT reuptake inhibitors as well as suggesting that 5-HT modulation per se is a potential therapeutic approach for inflammatory diseases.

Influence of estrous cycle hormonal fluctuations and gonadal hormones on the ventilatory response to hypoxia in female rats.

Sex hormones may influence many physiological processes. Recently, we demonstrated that hormonal fluctuations of cycling female rats do not affect respiratory parameters during hypercapnia. However, it is still unclear whether sex hormones and hormonal fluctuations that occur during the estrous cycle can affect breathing during a hypoxic challenge. Our study aimed to evaluate respiratory, metabolic, and thermal responses to hypoxia in female rats on different days of the estrous cycle (proestrus, estrus, metestrus, and diestrus) and in ovariectomized rats that received replacement with oil (OVX), estradiol (OVX + E2), or a combination of estradiol and progesterone (OVX + E2P). Ventilation (V E), tidal volume (V T), respiratory frequency (fR), oxygen consumption (VO2), and V E/VO2 were not different during the estrous cycle in normoxia or hypoxia. Body temperature (Tb) was higher during estrus, but decreased similarly in all groups during hypoxia. Compared with intact females in estrus, gonadectomized rats also had lower Tb in normoxia, but not in hypoxia. OVX rats experienced a significant drop in the ventilatory response to hypoxia, but hormonal replacement did not restore values to the levels of an intact animal. Our data demonstrate that the different phases of the estrous cycle do not alter ventilation during normoxia and hypoxia, but OVX animals display lower ventilatory responses to hypoxia compared with ovary-intact rats. Because estradiol and progesterone replacement did not cause significant differences in ventilation, our findings suggest that a yet-to-be-defined non-steroidal ovarian hormone is likely to stimulate the ventilatory responses to hypoxia in females.

Reproductive disorders in female rats after prenatal exposure to betamethasone.

Betamethasone is the drug of choice for antenatal treatment, promoting fetal lung maturation and decreasing mortality. Previous studies in rats reported male programming and alteration in sperm parameters and sexual behavior following intrauterine betamethasone exposure. The impact on the female reproductive development is not known. In this study, rat female offspring was assessed for sexual development, morphophysiology of the reproductive tract and fertility after maternal exposure to 0.1 mg kg-1 of betamethasone or vehicle on gestational days 12, 13, 18 and 19. The treatment promoted reduction of litter weight on postnatal day 1, morphological masculinization in females, delay in the age of puberty onset, reduction in estrus number, increase in estrous cycle length and increase in luteinizing hormone serum levels and uterus weight. The females from the betamethasone group showed an increase of myometrial uterine area and decrease in endometrial uterine area. These animals also performed less lordosis during the sexual behavior test and showed impaired reproductive performance. The uterus showed higher contraction in the treated group as shown by a pharmacological assay. In conclusion, prenatal betamethasone exposure in rats promoted female masculinization, altered sexual development and reproductive parameters. Copyright © 2017 John Wiley & Sons, Ltd.

Progesterone increased ß-endorphin innervation of the locus coeruleus, but ovarian steroids had no effect on noradrenergic neurodegeneration.

With the decline of ovarian steroids levels at menopause, many women experience an increase in anxiety and stress sensitivity. The locus coeruleus (LC), a central source of noradrenaline (NE), is activated by stress and is inhibited by ß-endorphin. Moreover, increased NE has been implicated in pathological anxiety syndromes. Hormone replacement therapy (HRT) in menopause appears to decrease anxiety and vulnerability to stress. Therefore, we questioned the effect of HRT on the inhibitory ß-endorphin innervation of the LC. In addition, we found that progesterone protects serotoninergic neurons in monkeys, leading us to question whether ovarian steroids are also neuroprotective in LC neurons in monkeys. Adult Rhesus monkeys (Macaca mulatta) were ovariectomized, and either treated with Silastic capsules that contained estradiol, estradiol+progesterone, progesterone alone or that were empty (ovariectomized; control). After 1month, the LC was obtained and processed for immunohistochemistry for ß-endorphin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL). The density of ß-endorphin axons was determined with image analysis using ImageJ. The TUNEL-positive neurons were counted in the entire LC. Progesterone-alone significantly increased the density of the ß-endorphin axons in the LC (p<0.01). No significant differences between groups in the number of TUNEL-positive cells in the LC were found. In conclusion, we found that HRT increases the inhibitory influence of ß-endorphin in the LC, which could, in turn, contribute to reduce anxiety and increase stress resilience. In addition, we did not find compelling evidence of neurodegeneration or neuroprotection by HRT in the LC of Rhesus monkeys.

Reproductive dysfunction after mercury exposure at low levels: evidence for a role of glutathione peroxidase (GPx) 1 and GPx4 in male rats.

Mercury is a ubiquitous environmental pollutant and mercury contamination and toxicity are serious hazards to human health. Some studies have shown that mercury impairs male reproductive function, but less is known about its effects following exposure at low doses and the possible mechanisms underlying its toxicity. Herein we show that exposure of rats to mercury chloride for 30 days (first dose 4.6µgkg-1, subsequent doses 0.07µgkg-1day-1) resulted in mean (±s.e.m.) blood mercury concentrations of 6.8±0.3ngmL-1, similar to that found in human blood after occupational exposure or released from removal of amalgam fillings. Even at these low concentrations, mercury was deposited in reproductive organs (testis, epididymis and prostate), impaired sperm membrane integrity, reduced the number of mature spermatozoa and, in the testes, promoted disorganisation, empty spaces and loss of germinal epithelium. Mercury increased levels of reactive oxygen species and the expression of glutathione peroxidase (GPx) 1 and GPx4. These results suggest that the toxic effects of mercury on the male reproductive system are due to its accumulation in reproductive organs and that the glutathione system is its potential target. The data also suggest, for the first time, a possible role of the selenoproteins GPx1 and GPx4 in the reproductive toxicity of mercury chloride.