On the role of sex steroids in biological functions by classical and non-classical pathways. An update.

The sex steroid hormones (SSHs) play several roles in regulation of various processes in the cardiovascular, immune, muscular and neural systems. SSHs affect prenatal and postnatal development of various brain structures, including regions associated with important physiological, behavioral, cognitive, and emotional functions. This action can be mediated by either intracellular or transmembrane receptors. While the classical mechanisms of SSHs action are relatively well examined, the physiological importance of non-classical mechanism of SSHs action through membrane-associated and transmembrane receptors in the brain remains unclear. The most recent summary describing the role of SSHs in different body systems is lacking. Therefore, the aim of this review is to discuss classical and non-classical signaling pathways of testosterone and estradiol action via their receptors at functional, cellular, tissue level and to describe the effects on various body systems and behavior. Particular emphasis will be on brain regions including the hippocampus, hypothalamus, frontal cortex and cerebellum.

The age-dependent effect of pre-pubertal castration on anxiety-like behaviour in male rats.

Adolescence is considered to be a critical period of sex hormone action (re)organising the brain and determining the behavioural phenotype. Such organisational effects in the brain might be the cause of sex differences in some behavioural features. In this experiment, we aimed to examine the role of pubertal sex hormones in development of anxiety in male rats. Male rats underwent gonadectomy prior to puberty onset, and were tested for explorative and anxiety-like behaviour in adolescence as well as in young adulthood. In adolescence, but not in adulthood, gonadectomised rats spend by 50% more time (p < .05) in the centre zone of the open-field than sham-operated counterparts. Young adult gonadectomised rats showed approximately 1.5-fold greater exploratory activity, in both open field (p < .001) and elevated plus maze (p < .01), in comparison with young adult control rats. Our results indicate that pre-pubertal castration may have test-specific anxiolytic effect in adolescent male rats, and it may attenuate the decline in explorative behaviour in young adult males. These differences in short- and long-term effects of gonadectomy could explain some contradictory results of previous studies on the role of testosterone in anxiety-like behaviour of male rodents. Thus, the age-specific consequences of pre-pubertal hormone deprivation should be considered.

Short-term effects of continuous positive airway pressure on oxidative stress in severe sleep apnea.

PURPOSE: Obstructive sleep apnea (OSA) is associated with oxidative stress that is involved in the pathogenesis of cardiovascular and metabolic complications. The concentrations of salivary markers of oxidative stress in patients with OSA increase considerably during the night. The dynamics is not affected by continuous positive airway pressure (CPAP) in mild to moderate OSA. The aim of this study was to analyze the short-term effects of CPAP on salivary oxidative stress markers in patients with severe OSA. METHODS: Salivary samples were collected from 24 patients with apnea-hypopnea index higher than 30 during the first (diagnostic) night, who were treated by CPAP during the second (therapeutic) night. RESULTS: The salivary markers of oxidative stress (TBARS, AGEs, and AOPP) were higher in the morning after the diagnostic night when compared to the evening concentrations (p < 0.01 for TBARS and p < 0.05 for AGEs and AOPP). Treatment by CPAP significantly decreased the morning concentrations of TBARS, AOPP (p < 0.01 for both), and AGEs (p < 0.05). Also, TBARS and AGEs positively correlated with apnea-hypopnea index (r = 0.48 and 0.49, respectively; p < 0.05). Antioxidant statuss was not affected. CONCLUSION: Severe OSA is associated with increased levels of saliva markers for lipid peroxidation, protein oxidative damage, and carbonyl damage. Even short-term CPAP partially prevents oxidative and carbonyl stress during the night and this can be monitored non-invasively using saliva.

17ß-Estradiol treatment reversed left ventricular dysfunction in castrated male rats: an echocardiographic study.

No data are available on heart function in chronic testosterone deficiency and on the effect of estrogen treatment. Eighteen 4-week-old male Lewis rats were randomly divided into 3 groups (n = 6): 1 group of sham-operated rats and 2 groups of castrated rats. Sixty-six weeks after surgery, 1 castrated group received a dose of 17ß-estradiol (10 µg/kg per day) and the remaining 2 groups received a placebo subcutaneously for 14 days. Left ventricular (LV) systolic and diastolic functions were measured by transthoracic echocardiography. Castration decreased LV ejection fraction (9%) and fractional shortening (15%) and deteriorated LV diastolic function (94%). 17ß-Estradiol treatment increased LV ejection fraction (15%) and fractional shortening (31%) and improved LV diastolic function (48%). Plasma testosterone concentrations were decreased in both castrated groups. In conclusion, chronic testosterone deficiency induced LV systolic and diastolic dysfunction; these disorders were reversed by short-term treatment with 17ß-estradiol.

Oxidative and carbonyl stress in pregnant women with obstructive sleep apnea.

PURPOSE: Pregnant women are particularly susceptible to sleep-disordered breathing. Obstructive sleep apnea (OSA) in pregnancy is associated with poor pregnancy and fetal outcomes. Oxidative stress caused by intermittent hypoxemia and reoxygenation may impact pregnancy health. We hypothesize that pregnant women with OSA have a pronounced oxidative stress profile. METHODS: A case-control study was performed to study oxidative stress markers in the serum of pregnant women with or without OSA. Patients with OSA were identified between 2003 and 2009. Contemporaneous controls were pregnant subjects without apnea, gasping, or snoring around the time of delivery. Serum markers of oxidative and carbonyl stress were measured by spectrophotometric/fluorometric methods. Multiple linear regression analysis was used with a model including age, body mass index at delivery, history of diabetes, and gestational age. RESULTS: Serum samples from 23 OSA cases and 41 controls were identified. Advanced oxidation protein products, a marker for oxidative stress, and advanced glycation end products (AGEs), a marker for carbonyl stress, were significantly lower in women with OSA than in controls (p value <0.0001). Total antioxidant capacity was higher in women with OSA in comparison to controls (p value <0.0001). The difference in AGEs remained significant even after adjusting for confounders. CONCLUSION: Contrary to our hypothesis, the results of this study suggest that pregnant women with OSA have higher antioxidant capacity and lower oxidative and carbonyl stress markers compared to controls, suggesting a possible protective effect of intermittent hypoxia. Whether OSA in pregnancy impacts oxidative stress differently than OSA in the general population remains to be confirmed.

Effect of melatonin on the behaviour of rats with continuous light-induced hypertension.

This study investigated whether continuous light exposure (CLE) results in behavioural disturbances in rats and whether melatonin can modify these potential changes. Four groups of 3-month-old Wistar rats were treated as follows for six weeks: control, melatonin, CLE, and CLE with melatonin. CLE increased systolic blood pressure and melatonin reduced it. No changes in behavioural patterns by CLE were observed. In the controls, melatonin reduced both exploration and locomotion but these parameters remained uninfluenced in the CLE. We conclude that melatonin exerted a different impact on behaviour in controls and in the CLE group.

Deoxyribonuclease partially ameliorates thioacetamide-induced hepatorenal injury.

Several recent studies have shown that liver injury is associated with the release of DNA from hepatocytes. This DNA stimulates innate immunity and induces sterile inflammation, exacerbating liver damage. Similar mechanisms have been described for acute renal injury. Deoxyribonuclease degrades cell-free DNA and can potentially prevent some of the induced tissue damage. This study analyzed the effects of thioacetamide-induced hepatorenal injury on plasma DNA in rats. Plasma DNA of both nuclear and mitochondrial origin was higher in thioacetamide-treated animals. Administration of deoxyribonuclease resulted in a mild, nonsignificant decrease in total plasma DNA and plasma DNA of mitochondrial origin but not of nuclear origin. This was accompanied by a decrease in bilirubin, creatinine, and blood urea nitrogen as markers of renal function. In conclusion, the study confirmed the hepatotoxic and nephrotoxic effect of thioacetamide. The associated increase in cell-free DNA seems to be involved in hepatorenal pathogenesis because treatment with deoxyribonuclease resulted in a partial prevention of hepatorenal injury. Further experiments will focus on the effects of long-term treatment with deoxyribonuclease in other clinically more relevant models. Clinical studies should test endogenous deoxyribonuclease activity as a potential risk determinant for kidney or liver failure.NEW & NOTEWORTHY Thioacetamide-induced hepatorenal injury resulted in higher plasma cell-free DNA. Deoxyribonuclease decreased average cell-free DNA of mitochondrial origin but not nuclear origin. Deoxyribonuclease partially prevented hepatorenal injury in rats.

Sex differences and sex hormones in anxiety-like behavior of aging rats.

Sex differences in the prevalence of affective disorders might be attributable to different sex hormone milieu. The effects of short-term sex hormone deficiency on behavior, especially on anxiety have been studied in numerous animal experiments, mainly on young adult rats and mice. However, sex differences in aged animals and the effects of long-term hypogonadism are understudied. The aim of our study was to analyze sex differences in anxiety-like behavior in aged rats and to prove whether they can be attributed to endogenous sex hormone production in males. A battery of tests was performed to assess anxiety-like behavior in aged female, male and gonadectomized male rats castrated before puberty. In addition, the aged gonadectomized male rats were treated with a single injection of estradiol or testosterone or supplemented with estradiol for two-weeks. Female rats displayed a less anxious behavior than male rats in most of the conducted behavioral tests except the light-dark box. Long-term androgen deficiency decreased the sex difference in anxiety either partially (open field, PhenoTyper cage) or completely (elevated plus maze). Neither single injection of sex hormones, nor two-week supplementation of estradiol in gonadectomized aged male rats significantly affected their anxiety-like behavior in the elevated plus maze. In conclusion, our results confirm sex differences in anxiety in aged rats likely mediated by endogenous testosterone production in males. Whether long-term supplementation with exogenous sex hormones could affect anxiety-like behavior in elderly individuals remains to be elucidated.

Sex-dependent effects of letrozole on anxiety in middle-aged rats.

Aromatase catalyzes the conversion of testosterone to estradiol and is involved in the physiological effects of sex hormones on brain function. Animal experiments have shown that the aromatase inhibitor, letrozole, can induce anxiety in young ovariectomized females that are used as a model of aging. Whether or not these effects would be similar in intact middle-aged animals is unknown. The aim of our study was to analyze the effects of letrozole on anxiety in middle-aged rats of both sexes. Fifteen month old male and female rats were treated daily with either letrozole or vehicle for 2 weeks. The elevated plus maze was used to test anxiety-like behaviour. Sex differences were found not only in plasma concentrations of testosterone but also in the effects of letrozole treatment on plasma testosterone (P<.05). The interaction between sex and treatment was also proven in locomotor activity (P<.05) and time spent in the open arms of the elevated plus maze (P<.05). Letrozole-treated male rats spent 95% less time in the open arms of the elevated plus maze than the control rats did (P<.05) suggesting an anxiogenic effect of aromatase inhibition. This difference was not found between letrozole-treated and vehicle-treated females. In contrast to previous experiments on young animals, letrozole seems to induce anxiety in male but not in female middle-aged rats. This sex-specific effect might be related to sex differences of oestrogen and androgen signalling in aging brains. These results should be taken into account in clinical applications of letrozole, especially in men.

The effects of anthocyanin-rich wheat diet on the oxidative status and behavior of rats.

AIM: To evaluate the effect of food containing anthocyanin-rich wheat on oxidative status and behavior of healthy rats. METHODS: Twenty male rats were divided into the control and anthocyanin group. Oral glucose tolerance test was performed, and proteinuria and creatinine clearance were measured. Behavioral analysis was performed in Phenotyper cages. Serum and tissues were collected to measure the markers of oxidative stress and antioxidant status. RESULTS: Anthocyanins significantly increased total antioxidant capacity in serum (P=0.039), decreased advanced oxidation protein products in the kidney (P=0.002), but increased thiobarbituric acid reactive substances in the kidney compared to the control group. No significant difference between the groups was found in the markers of oxidative stress in the liver and colon, as well as in renal functions and glucose metabolism. The anthocyanin group spent significantly less time in the spotlight zone of the Phenotyper cages (P=0.040), indicating higher anxiety-like behavior. CONCLUSION: Food containing anthocyanin-rich wheat had positive effects on serum antioxidant status and kidney protein oxidation, but increased lipid peroxidation in the kidney and modified animal behavior related to anxiety. The molecular mechanisms leading to observed effects should be further elucidated.

Intracerebroventricular oxytocin administration in rats enhances object recognition and increases expression of neurotrophins, microtubule-associated protein 2, and synapsin I.

Brain oxytocin regulates a variety of social and affiliative behaviors and affects also learning and memory. However, mechanisms of its action at the level of neuronal circuits are not fully understood. The present study tests the hypothesis that molecular factors required for memory formation and synaptic plasticity, including brain-derived neurotrophic factor, neural growth factor, nestin, microtubule-associated protein 2 (MAP2), and synapsin I, are enhanced by central administration of oxytocin. We also investigated whether oxytocin enhances object recognition and acts as anxiolytic agent. Therefore, male Wistar rats were infused continuously with oxytocin (20 ng/µl) via an osmotic minipump into the lateral cerebral ventricle for 7 days; controls were infused with vehicle. The object recognition test, open field test, and elevated plus maze test were performed on the sixth, seventh, and eighth days from starting the infusion. No significant effects of oxytocin on anxious-like behavior were observed. The object recognition test showed that oxytocin-treated rats significantly preferred unknown objects. Oxytocin treatment significantly increased gene expression and protein levels of neurotrophins, MAP2, and synapsin I in the hippocampus. No changes were observed in nestin expression. Our results provide the first direct evidence implicating oxytocin as a regulator of brain plasticity at the level of changes of neuronal growth factors, cytoskeletal proteins, and behavior. The data support assumption that oxytocin is important for short-term hippocampus-dependent memory.

The renal effects of prenatal testosterone in rats.

PURPOSE: Previous studies have shown that prenatal testosterone affects the development of not only reproductive organs but also the brain and even glucose metabolism. Whether prenatal testosterone influences the kidney development is largely unknown. We analyzed whether testosterone modulation during prenatal development would affect renal function and the number of nephrons in adult offspring. MATERIALS AND METHODS: Pregnant rats were treated with olive oil, testosterone (2 mg/kg), the androgen receptor blocker flutamide (5 mg/kg) or testosterone plus flutamide via daily intramuscular injections from gestation day 14 until delivery. Renal histology and functional parameters were assessed in male and female adult offspring. Macerated kidneys were used for nephron counting. RESULTS: Prenatal testosterone administration increased proteinuria in male rats by 256%. A similar 134% effect in female rats was not statistically significant. This effect was prevented when flutamide was co-administered. In male rats prenatal testosterone increased blood urea nitrogen. In female rats flutamide increased creatinine clearance. In male rats prenatal testosterone and flutamide led to higher and lower, respectively, interstitial collagen deposition in adulthood. CONCLUSIONS: Prenatal testosterone induces proteinuria in adulthood. This effect is mediated via androgen receptor. Additional effects seem to be sex specific. Further studies should focus on the timing and dosing of testosterone as well as the applicability to human development.

Single consumption of Bryndza cheese temporarily affects oral microbiota and salivary markers of oxidative stress.

Several oral diseases are associated with changes in oral microbiota and higher oxidative stress. Enterococcus faecalis has been hypothesized to directly contribute to the oxidative stress in oral cavity. The aim of this study was to examine the effect of single consumption of unpasteurized Bryndza cheese containing enterococci on changes of microbiota and oxidative status in saliva. Fourteen healthy volunteers aged 23-30 years were asked to eat 100 g of Bryndza cheese. Saliva samples were collected before and 1, 10, 100 min, and 24 h after Bryndza cheese consumption. Species-specific PCR and terminal restriction fragment length polymorphism (T-RFLP) analysis were used to characterize oral microbiota. Markers of oxidative stress and antioxidant status were measured in saliva. PCR identified E. faecium in 36 % of probands saliva up to 1 day after consumption of enterococci containing Bryndza cheese. E. faecalis was detected in 57 % of probands saliva up to 10 min and in one proband up to 100 min after Bryndza cheese consumption. T-RFLP analysis confirmed short-term changes in composition of oral microbiota after Bryndza cheese ingestion. Nevertheless, the microbiota was completely restored after 24 h. One minute after ingestion of Bryndza cheese, salivary advanced oxidation protein products were significantly increased (by 74.6 %, P < 0.001), and total antioxidant capacity was decreased (by 22.0 %, P < 0.05). This study shows that single consumption of enterococci containing Bryndza cheese can temporally affect the composition of oral microbiota and oxidative stress in saliva. Further studies should identify the impact of these changes to the pathogenesis of oral diseases.

Salivary markers of oxidative stress in patients with obstructive sleep apnea treated with continuous positive airway pressure.

PURPOSE: Obstructive sleep apnea syndrome (OSAS) is characterized by elevated oxidative stress. Measurement of oxidative stress in saliva seems to be promising in long-term treatment monitoring of OSAS patients. In this study, our aim was to investigate whether short-term continuous positive airway pressure (CPAP) treatment would influence oxidative stress in saliva. METHODS: Patients with diagnosed OSAS (16 women, 28 men) underwent polysomnography during the first night and CPAP treatment during the second night. Saliva samples were taken in the evening and morning on both days. Markers of oxidative stress and antioxidant status were analyzed in saliva. RESULTS: Evening concentrations of the salivary thiobarbituric acid reacting substances (p < 0.001), advanced glycation end-products (p < 0.001), and advanced oxidation protein products (p < 0.01) were significantly lower than morning values during the diagnostic night. However, salivary concentrations of none of the oxidative stress markers were significantly influenced by the CPAP treatment. No changes in salivary antioxidant status after CPAP therapy were found. CONCLUSION: Salivary markers of oxidative stress and antioxidant status do not change significantly after one night treatment with CPAP. On the contrary, after 1 month with CPAP therapy, reduced markers of oxidative stress were reported. Therefore, the future studies should be focused on finding the optimal sampling frequency to clarify the potential of saliva for the monitoring of OSAS treatment.

Vanishing twin as a potential source of bias in non-invasive fetal sex determination: a case report.

Detection of fetal sex based on fetal DNA present in maternal plasma is already in clinical use. Here we present a case of false positivity during the first trimester which may be attributable to a vanishing twin. The presence of Y-chromosome-specific sequences is used as a marker to indicate a male fetus and the absence of a female fetus. Fetal sex determination was conducted in a pregnant woman at gestational week 10. The sample was positive in all triplicates. Ultrasonography at gestational week 20 revealed female sex. Analysis of sample taken at gestational week 22 indicated a female fetus. According to recently published meta-analyses, non-invasive prenatal diagnosis of fetal sex has high sensitivity and specificity values. Nevertheless, false negative and false positive cases occur. Future studies focusing on the dynamics of fetal DNA are needed. Vanishing twin might be one of the possible causes of false positivity in fetal sex determination.

Mitochondrial DNA variability and Covid-19 in the Slovak population.

Recent studies have shown that mitochondria are involved in the pathogenesis of Covid-19. Mitochondria play a role in production of reactive oxygen species and induction of an innate immune response, both important during infections. Common variability of mitochondrial DNA (mtDNA) can affect oxidative phosphorylation and the risk or lethality of cardiovascular, neurodegenerative diseases and sepsis. However, it is unclear whether susceptibility of severe Covid-19 might be affected by mtDNA variation. Thus, we have analyzed mtDNA in a sample of 446 Slovak patients hospitalized due to Covid-19 and a control population group consisting of 1874 individuals. MtDNA variants in the HVRI region have been analyzed and classified into haplogroups at various phylogenetic levels. Binary logistic regression was used to assess the risk of Covid-19. Haplogroups T1, H11, K and variants 16256C > T, 16265A > C, 16293A > G, 16311 T > C and 16399A > G were associated with an increased Covid-19 risk. On contrary, Haplogroup J1, haplogroup clusters H + U5b and T2b + U5b, and the mtDNA variant 16189 T > C were associated with decreased risk of Covid-19. Following the application of the Bonferroni correction, statistical significance was observed exclusively for the cluster of haplogroups H + U5b. Unsurprisingly, the most significant factor contributing to the mortality of patients with Covid-19 is the age of patients. Our findings suggest that mtDNA haplogroups can play a role in Covid-19 pathogenesis, thus potentially useful in identifying susceptibility to its severe form. To confirm these associations, further studies taking into account the nuclear genome or other non-biological influences are needed.

The effect of testosterone on the formation of brain structures.

It has been confirmed in several studies that testosterone can significantly affect brain development. Following metabolism of this hormone by 5α-reductase to dihydrotestosterone, testosterone may act via androgen receptors, or after conversion by aromatase to estradiol, it may act via estrogen receptors. The parts of the brain which are changed under the influence of sex hormones are known as sexually dimorphic nuclei, especially in the preoptic area of the hypothalamus. Nevertheless, evidence suggests that testosterone also influences the structure of the hippocampus, specifically CA1 and CA3 areas of the hippocampus, as well as the amygdala. These brain areas are designed to convert information from short-term into long-term memory. In this review, we summarize the effects of testosterone on the organization of brain structures with respect to spatial cognitive abilities in small rodents.

Antioxidant vitamins prevent oxidative and carbonyl stress in an animal model of obstructive sleep apnea.

PURPOSE: The aim of our study was to analyze the effects of an antioxidant treatment on markers of oxidative and carbonyl stress in a rat model of obstructive sleep apnea. METHODS: Wistar rats were randomized into six groups-according to gender and intervention-sham, intermittent hypoxia, and intermittent hypoxia with treatment by vitamins C and E. Rats underwent tracheostomy. The tracheal cannula was closed for 12 s every minute for 1 h to simulate obstructive sleep apnea-related intermittent hypoxia. In the treatment group, rats received vitamin C and E 24 h prior to surgery. RESULTS: The intervention had a significant effect on advanced oxidation protein products (p = 0.008) and advanced glycation end products-specific fluorescence (p = 0.006) but no effect on malondialdehyde. Oxidation and glycation protein products were higher in intermittent hypoxia groups than in sham and in treated groups. CONCLUSIONS: Antioxidants alleviate oxidative and carbonyl stress in an experimental model of obstructive sleep apnea. Future studies will show whether such treatment has any clinical value regarding cardiovascular complications of sleep apnea syndrome, preferably in patients with low compliance to continuous positive airway pressure.

Sex differences of oxidative stress markers in young healthy subjects are marker-specific in plasma but not in saliva.

BACKGROUND: Several studies showed there are sex differences in oxidative stress. An observational study analysing oxidative stress markers in young healthy men and women is lacking. Moreover, it is unclear whether the differences are related to sex hormones. AIM: The primary goal was to analyse differences in oxidative stress markers with regard to sex in plasma of young healthy subjects and whether differences are related to sex hormones. The secondary study compared oxidative stress markers in plasma with salivary samples. METHODS: Plasma and saliva samples were analysed from 158 young healthy probands. Established spectro-photometric/fluorometric methods were used to quantify oxidative stress markers. Sex hormones were measured using ELISA kits. RESULTS: In plasma, malondialdehyde and advanced glycation end products were significantly higher in women. Advanced oxidation protein products and the ferric reducing ability of plasma were higher in men. Sex hormones were not associated with oxidative stress markers. In saliva, analysed markers of antioxidant status were higher in men, but no sex differences were found in other markers. CONCLUSION: Observed parameters showed marker-specific sex differences in plasma, but these differences were not related to sex hormones. Plasma and saliva concentrations of biomarkers did not correlate, reflecting oral but not systemic conditions.