Locally Synthetized 17-ß-Estradiol Reverses Amyloid-ß-42-Induced Hippocampal Long-Term Potentiation Deficits.

Amyloid beta 1-42 (Aß42) aggregates acutely impair hippocampal long-term potentiation (LTP) of synaptic transmission, and 17ß-estradiol is crucial for hippocampal LTP. We tested whether boosting the synthesis of neural-derived 17ß-estradiol (nE2) saves hippocampal LTP by the neurotoxic action of Aß42. Electrophysiological recordings were performed to measure dentate gyrus (DG) LTP in rat hippocampal slices. Using a pharmacological approach, we tested the ability of nE2 to counteract the LTP impairment caused by acute exposure to soluble Aß42 aggregates. nE2 was found to be required for LTP in DG under physiological conditions. Blockade of steroid 5α-reductase with finasteride, by increasing nE2 synthesis from testosterone (T), completely recovered LTP in slices treated with soluble Aß42 aggregates. Modulation of the glutamate N-methyl-D aspartate receptor (NMDAR) by memantine effectively rescued the LTP deficit observed in slices exposed to Aß42, and memantine prevented LTP reduction observed under the blocking of nE2 synthesis. nE2 is able to counteract Aß42-induced synaptic dysfunction. This effect depends on a rapid, non-genomic mechanism of action of nE2, which may share a common pathway with glutamate NMDAR signaling.

Kisspeptin modulation of nonapeptide and cytochrome P450 aromatase mRNA expression in the brain and ovary of the catfish Heteropneustes fossilis: in vivo and in vitro studies.

In Heteropneustes fossilis, kisspeptins (Kiss) and nonapeptides (NPs; vasotocin, Vt; isotocin, Itb; Val8-isotocin, Ita) stimulate the hypothalamus-pituitary-gonadal (HPG) axis, and estrogen feedback modulates the expression of these systems. In this study, functional interactions among these regulatory systems were demonstrated in the brain and ovary at the mRNA expression level. Human KISS1 (hKISS1) and H. fossilis Kiss2 (HfKiss2) produced biphasic effects on brain and ovarian vt, itb and ita expression at 24 h post injection: low and median doses produced inhibition, no change or mild stimulation, and the highest dose consistently stimulated the mRNA levels. The Kiss peptides produced an upregulation of NP mRNA expression at 24 h incubation of brain and ovarian slices by increasing the concentration of hKISS1 and HfKiss2. The kiss peptides stimulated brain cyp19a1b and ovary cyp19a1a expression, both in vivo and in vitro. Peptide234, a Kiss1 receptor antagonist, inhibited basal mRNA expression of the NPs, cyp19a1b and cyp19a1a, which was prevented by the Kiss peptides, both in vivo and in vitro. In all the experiments, HfKiss2 was more effective than hKISS1 in modulating mRNA expression. The results suggest that the NP and E2 systems are functional targets of Kiss peptides and interact with each other.

Genotoxicity and endocrine disruption potential of haloacetic acids in human placental and lung cells.

Chlorination of water results in the formation of haloacetic acids (HAAs) as major disinfection byproducts (DBPs). Previous studies have reported some HAAs species to act as cytotoxic, genotoxic, and carcinogenic. This work aimed at further exploring the toxicity potential of the most investigated HAAs (chloroacetic (CAA), bromoacetic (BAA), iodoacetic (IAA) acid) and HAAs species with high content of bromine (tribromoacetic acid (TBAA)), and iodine in their structures (chloroiodoacetic (CIAA) and diiodoacetic acid (DIAA)) to human cells. Novel knowledge was generated regarding cytotoxicity, oxidative stress, endocrine disrupting potential, and genotoxicity of these HAAs by using human placental and lung cells as in vitro models, not previously used for DBP assessment. IAA showed the highest cytotoxicity (EC50: 7.5 µM) and ability to generate ROS (up to 3-fold) in placental cells, followed by BAA (EC50: 20-25 µM and 2.1-fold). TBAA, CAA, DIAA, and CIAA showed no significant cytotoxicity (EC50 > 250 µM). All tested HAAs decreased the expression of the steroidogenic gene hsd17b1 up to 40 % in placental cells, and IAA and BAA (0.01-1 µM) slightly inhibited the aromatase activity. HAAs also induced the formation of micronuclei in A549 lung cells after 48 h of exposure. IAA and BAA showed a non-significant increase in micronuclei formation at low concentrations (1 µM), while BAA, CAA, CIAA and TBAA were genotoxic at exposure concentrations above 10 µM (100 µM in the case of DIAA). These results point to genotoxic and endocrine disruption effects associated with HAA exposure at low concentrations (0.01-1 µM), and the usefulness of the selected bioassays to provide fast and sensitive responses to HAA exposure, particularly in terms of genotoxicity and endocrine disruption effects. Further studies are needed to define thresholds that better protect public health.

Genetic polymorphisms related to muscular strength and flexibility are associated with artistic gymnastic performance in the Japanese population.

This study aimed to examine how genetic polymorphisms related to muscular strength and flexibility influence artistic gymnastic performance in an attempt to identify a novel polymorphism associated with flexibility. In study 1, the passive straight-leg-raise (PSLR) score and aromatase gene CYP19A1 rs936306 polymorphism, a key enzyme for estrogen biosynthesis, were assessed in 278 individuals. In study 2, athletes (281 gymnasts and 1908 other athletes) were asked about their competition level, and gymnasts were assessed using the difficulty score (D-score) for each event. Muscular strength- (ACTN3 R577X rs1815739 and ACE I/D rs4341) and flexibility-related (ESR1 rs2234693 T/C and CYP19A1 rs936306 C/T) genetic polymorphisms were analyzed. In study 1, males with the CYP19A1 CT + TT genotype showed significantly higher PSLR scores than those with the CC genotype. In study 2, male gymnasts with the R allele of ACTN3 R577X showed a correlation with the floor, rings, vault, and total D-scores. In addition, male gymnasts with the C allele of ESR1 T/C and T allele of CYP19A1 C/T polymorphisms were correlated with the pommel horse, parallel bars, horizontal bar, and total D-scores. Furthermore, genotype scores of these three polymorphisms correlated with the total D-scores and competition levels in male gymnasts. In contrast, no such associations were observed in female gymnasts. Our findings suggest that muscular strength- and flexibility-related polymorphisms play important roles in achieving high performance in male artistic gymnastics by specifically influencing the performance of events that require muscular strength and flexibility, respectively.HighlightsEstrogen-related CYP19A1 polymorphism is a novel determinant of flexibility in males.Muscular strength- and flexibility-related polymorphisms play important roles in high performance in male artistic gymnastics.Genotypes of ACTN3 R577X, ESR1 rs2234693, and CYP19A1 rs936306 may contribute to training plan optimization and event selection in artistic gymnastics.

Genetic polymorphisms in CYP19A1 and ESR1 are associated with serum CK activity after prolonged running in men.

This study aimed to clarify 1) the influence of genetic polymorphisms in the cytochrome P450 aromatase gene (CYP19A1) on circulating estradiol levels in men and 2) whether estrogen-related genetic polymorphisms, such as the CYP19A1 rs936306 and estrogen receptor-α (ESR1) rs2234693 polymorphisms, predict exercise-induced serum creatine kinase (CK) activity, which is an index of skeletal muscle membrane disruption. Serum estradiol levels were examined in young men (n = 167). In a different cohort, serum CK activity was analyzed in a 2-day ultramarathon race: baseline, after the first day, and after the second day (114 males and 25 females). Genetic polymorphisms in CYP19A1 rs936306 C/T and ESR1 rs2234693 T/C were analyzed using the TaqMan SNP Genotyping Assay. Male subjects with the TT genotype of the CYP19A1 polymorphism exhibited significantly higher serum estradiol levels than the C allele carriers. Male runners had significantly higher postrace serum CK activity than female runners. The change in the CK activity during the ultramarathon race was significantly lower in male subjects with the CYP19A1 TT genotype than in those with the CC + CT genotypes and was correlated with the number of C alleles in ESR1 rs2234693 in male subjects. Furthermore, the genotype scores of these two polymorphisms were significantly correlated with changes in serum CK activity during race (r = -0.279, P = 0.003). The results of this study suggest that genetic polymorphisms in CYP19A1 rs936306 influence serum estradiol levels in men, and genetic polymorphisms in CYP19A1 and ESR1 are associated with serum CK activity in men.NEW & NOTEWORTHY Men with the TT genotype of the CYP19A1 polymorphism exhibited higher circulating estradiol levels than the TC + CC genotype. The TT genotype in the CYP19A1 polymorphism and the C allele of the ESR1 polymorphism, an allele increasing ESR1 expression, were associated with low serum CK activity after the ultramarathon. A combination of these polymorphisms was correlated with changes in the serum CK activity. Therefore, estrogen-related genetic polymorphisms partially predict exercise-induced muscle damage, that is, skeletal muscle membrane disruption.

The genome of New Zealand trevally (Carangidae: Pseudocaranx georgianus) uncovers a XY sex determination locus.

BACKGROUND: The genetic control of sex determination in teleost species is poorly understood. This is partly because of the diversity of mechanisms that determine sex in this large group of vertebrates, including constitutive genes linked to sex chromosomes, polygenic constitutive mechanisms, environmental factors, hermaphroditism, and unisexuality. Here we use a de novo genome assembly of New Zealand silver trevally (Pseudocaranx georgianus) together with sex-specific whole genome sequencing data to detect sexually divergent genomic regions, identify candidate genes and develop molecular makers. RESULTS: The de novo assembly of an unsexed trevally (Trevally_v1) resulted in a final assembly of 579.4 Mb in length, with a N50 of 25.2 Mb. Of the assembled scaffolds, 24 were of chromosome scale, ranging from 11 to 31 Mb in length. A total of 28,416 genes were annotated after 12.8 % of the assembly was masked with repetitive elements. Whole genome re-sequencing of 13 wild sexed trevally (seven males and six females) identified two sexually divergent regions located on two scaffolds, including a 6 kb region at the proximal end of chromosome 21. Blast analyses revealed similarity between one region and the aromatase genes cyp19 (a1a/b) (E-value < 1.00E-25, identity > 78.8 %). Males contained higher numbers of heterozygous variants in both regions, while females showed regions of very low read-depth, indicative of male-specificity of this genomic region. Molecular markers were developed and subsequently tested on 96 histologically-sexed fish (42 males and 54 females). Three markers amplified in absolute correspondence with sex (positive in males, negative in females). CONCLUSIONS: The higher number of heterozygous variants in males combined with the absence of these regions in females support a XY sex-determination model, indicating that the trevally_v1 genome assembly was developed from a male specimen. This sex system contrasts with the ZW sex-determination model documented in closely related carangid species. Our results indicate a sex-determining function of a cyp19a1a-like gene, suggesting the molecular pathway of sex determination is somewhat conserved in this family. The genomic resources developed here will facilitate future comparative work, and enable improved insights into the varied sex determination pathways in teleosts. The sex marker developed in this study will be a valuable resource for aquaculture selective breeding programmes, and for determining sex ratios in wild populations.

The Aromatase-Estrogen System in the Testes of Non-Mammalian Vertebrates.

Estrogens are important physiological regulators of testicular activity in vertebrates. Estrogen levels depend on the activity of P450 aromatase, the enzyme responsible for the irreversible conversion of testosterone into 17ß-estradiol. Therefore, P450 aromatase is the key player in the aromatase-estrogen system. The present review offers a comparative overview of P450 aromatase activity in male gonads of amphibians, reptiles, and birds, with a particular emphasis on the functions of the aromatase-estrogen system in these organisms during their developmental and adult stages. The aromatase-estrogen system appears to be crucial for the sex differentiation of gonads in vertebrates. Administration of aromatase inhibitors prior to sexual differentiation of gonads results in the development of males rather than females. In adults, both aromatase and estrogen receptors are expressed in somatic cells, Leydig and Sertoli cells, as well as germ cells, with certain differences among different species. In seasonal breeding species, the aromatase-estrogen system serves as an "on/off" switch for spermatogenesis. In some amphibian and reptilian species, increased estrogen levels in post-reproductive testes are responsible for blocking spermatogenesis, whereas, in some species of birds, estrogens function synergistically with testosterone to promote spermatogenesis. Recent evidence indicates that the production of the aromatase enzyme in excessive amounts reduces the reproductive performance in avian species of commercial interest. The use of aromatase inhibitors to improve fertility has yielded suitable positive results. Therefore, it appears that the role of the aromatase-estrogen system in regulating the testicular activity differs not only among the different classes of vertebrates but also among different species within the same class.

11-Oxygenated Estrogens Are a Novel Class of Human Estrogens but Do not Contribute to the Circulating Estrogen Pool.

Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen both in men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we use steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in 3 different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11ß-hydroxy-17ß-estradiol binds and activates estrogen receptors α and ß and that 11ß-hydroxy-17ß-estradiol and the classic androgen pathway-derived active estrogen, 17ß-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data show that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.

Rapid Estrogenic and Androgenic Neurosteroids Effects in the Induction of Long-Term Synaptic Changes: Implication for Early Memory Formation.

Mounting experimental evidence demonstrate that sex neuroactive steroids (neurosteroids) are essential for memory formation. Neurosteroids have a profound impact on the function and structure of neural circuits and their local synthesis is necessary for the induction of both long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission and for neural spine formation in different areas of the central nervous system (CNS). Several studies demonstrated that in the hippocampus, 17ß-estradiol (E2) is necessary for inducing LTP, while 5α-dihydrotestosterone (DHT) is necessary for inducing LTD. This contribution has been proven by administering sex neurosteroids in rodent models and by using blocking agents of their synthesis or of their specific receptors. The general opposite role of sex neurosteroids in synaptic plasticity appears to be dependent on their different local availability in response to low or high frequency of synaptic stimulation, allowing the induction of bidirectional synaptic plasticity. The relevant contribution of these neurosteroids to synaptic plasticity has also been described in other brain regions involved in memory processes such as motor learning, as in the case of the vestibular nuclei, the cerebellum, and the basal ganglia, or as the emotional circuit of the amygdala. The rapid effects of sex neurosteroids on neural synaptic plasticity need the maintenance of a tonic or phasic local steroid synthesis determined by neural activity but might also be influenced by circulating hormones, age, and gender. To disclose the exact mechanisms how sex neurosteroids participate in finely tuning long-term synaptic changes and spine remodeling, further investigation is required.

Mild Exercise Rescues Steroidogenesis and Spermatogenesis in Rats Submitted to Food Withdrawal.

The present investigation was undertaken to increase our insight into the molecular basis of the physiological changes in rat testis induced by food withdrawal, and to clarify whether reduced testicular function can be ameliorated by mild exercise. Male rats were selected for four separate experiments. The first of each group was chow-fed, the second was chow-fed and submitted to exercise (5 bouts in total for 30 min at 15 m/min, and 0° inclination), the third was submitted to food withdrawal (66 h) and the fourth was submitted to food withdrawal and to exercise. At the end of experiments, we investigated (i) serum and testicular sex hormone levels; (ii) protein levels of StAR, 3ß-Hydroxysteroid dehydrogenase (3ß-HSD) and P450 aromatase, which play a key role in steroid hormone biosynthesis; and (iii) protein levels of mitotic and meiotic markers of spermatogenesis in rats, in relation to testis morphology and morphometry. We found that mild exercise or food withdrawal alone induced a significant increase or decrease in both serum and testis testosterone levels, respectively. Interestingly, we found that these levels were brought back to basal levels when food withdrawal was combined with mild exercise. The changes in testosterone levels observed in our experimental groups correlated well with the expression of steroidogenic enzymes as well as with spermatogenic activity. With mild exercise the increased testosterone/17ß-estradiol (T/E2) ratio in the testis correlated with an increased spermatogenic activity. The T/E2 ratio dropped in fasted rats and was significantly reversed when food withdrawal was combined with exercise. Histological and morphometric analyses confirmed that spermatogenic activity varied in concomitance with each experimental condition. Importantly, the testis and serum T/E2 ratios correlated, confirming that exercise rescues the decline in food withdrawal-induced spermatogenesis. In conclusion, this study highlights that mild exercise normalizes the reduced spermatogenic activity caused by food withdrawal through the modulation of the steroidogenic pathway and restoring the T/E2 ratio, underlining the beneficial effects of mild exercise on the prevention and/or amelioration of reduced testis function caused by restricted caloric intake.

Detection of endometriosis using immunocytochemistry of P450 Aromatase expressions in eutopic endometrial cells obtained from menstrual sloughing: a diagnostic study.

OBJECTIVE: To explore the possibility of a new diagnostic approach of endometriosis based on immunocytochemistry scoring of Aromatase P450 expressions in endometrial cells collected from menstrual sloughing. This is a case control study. Immuncytochemistry scores vs. histopathological examination in one tertiary- and secondary-level hospital in Bandung; two secondary level hospital in Garut and Sumedang, West Java, Indonesia. Thirty-five patients with and without endometriosis were enrolled. All subjects had diagnostic procedures for endometriosis suspicion, with addition menstrual blood samples for cytopathological examination. The specimens were sent for immunocytochemistry assessment of P450 Aromatase expressions (ICAPEC). The previous procedure resulted in cut-off point of histo score (H-score), sensitivity, specificity, (+) and (-) ICAPEC predictive value. RESULTS: The P450 Aromatase expression in endometrial cells of women with endometriosis was significantly stronger than without one. The cut-off point of H-scores to detect endometriosis was > 4. By this criteria, H-score had 94.6% sensitivity, 90.9% specificity, 92% positive predictive value and 93% negative predictive value. Immunocytochemistry scoring of Aromatase P450 expression in endometrial cells (ICAPEC) derived from menstrual blood specimen was a good candidate as alternatives approach in diagnostic procedure of endometriosis. Application and evaluation in clinical practice would provide the economically benefit in diagnostic procedure.

The regulatory effect of genistein on P450 aromatase and follicle-stimulating hormone receptor in mouse experimental model of menopausal metabolic syndrome.

In order to investigate the mechanism of genistein (Gen) in the treatment of climacteric syndrome, an in vivo study was performed to investigate the beneficial effects of genistein on the expression of P450 aromatase (P450 arom) and follicle-stimulating hormone receptor (FSHR) in the mouse ovary and uterus. Fifty female ICR mice (45 ± 5g, n = 50), aged 12 months, were divided into the following five groups with 10 animals in each: blank control group (CG), low-dose genistein group (L-Gen), middle-dose genistein group (M-Gen) and high-dose genistein group (H-Gen) (received 15, 30 and 60 mg/kg of genistein, respectively), and oestrogen group (EG; received 0.5 mg/kg diethylstilbestrol). The expression levels of the FSHR protein were determined by an immunohistochemical staining method. The expression of P450 arom, Cytochrome P450 19 (CYP19) and FSHR was quantified by real-time PCR. Immunohistochemical results showed that the expression levels of the FSHR protein in the M-Gen (average stained area: 20.79) and the H-Gen (average stained area: 21.21) groups were significantly stronger than in the CG (average area was 17.24) group (p < .05). The expression levels of CYP19 mRNA and P450 arom were positively correlated with the dose of genistein. Specifically, the relative expression levels in the H-Gen and EG groups were more than 1.5 times higher than in the CG group (p < .05). Genistein played a significant role in regulating aromatase and FSHR gene expression to improve perimenopausal ovarian and uterine function.

Effect of genistein on the gene expressions of androgen generating key enzymes StAR, P450scc and CYP19 in rat ovary.

In this investigation, the effects of genistein (GEN) on the expression of steroidogenic genes such as steroidogenic acute regulatory protein (StAR), side-chain cleavage enzymes (P450scc) and cytochrome P450 aromatase (CYP19) were assessed. For this study, forty young female Sprague Dawley (SD) rats at aged 2-3 months (200±20 g) and forty aged female SD rats aged 10-12 months (490±20 g) were selected. Also, based on weight they were divided into a negative control group (NC), three different GEN dose groups, which received GEN of 15, 30, 60 mg/kg, and a positive control group (PC). The experiment lasted 30 days. Concentrations of serum hormones were determined by Enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions of StAR, P450scc and CYP19 were determined by Real-Time PCR and western blot techniques. It was observed that 30-60 mg/kg GEN could increase the expression of androgen generating key enzymes in the young rat ovary. GEN also significantly increased progesterone and E2 levels in the serum of aged rats and reduced the levels of LH and FSH in the serum of both young and aged rats. Compared with young rats, the effect of GEN on the ovary of aged rats was stronger and a lower dose of GEN (15 mg/kg) showed an obvious effect on these indicators. GEN influenced both estrogen level and indicators associated with estrogen and androgen transformation processes, which indicates that GEN can impair the growth and maturation of the ovary.

Placental ESRRG-CYP19A1 Expressions and Circulating 17-Beta Estradiol in IUGR Pregnancies.

Introduction: Sex steroids are regulating factors for intrauterine growth. 17-ß Estradiol (E2) is particularly critical to a physiological pregnancy, as increased maternal E2 was correlated to lower fetal weight at delivery. The placenta itself is a primary source of estrogens, synthetized from cholesterol precursors. Cytochrome P450 aromatase (encoded by CYP19A1 gene) is a rate-limiting enzyme for E2 biosynthesis. CYP19A1 transcription is supported by Estrogen Related-Receptor Gamma (ERRγ- ESRRG gene), which thus has an indirect role in placental steroidogenesis. Here we investigated maternal E2 levels and placental CYP19A1 and ESRRG expressions in pregnancies with IntraUterine Growth Restriction (IUGR). Methods: Singleton pregnancies were studied. E2 was measured in maternal plasma by electrochemiluminescence in 16 term controls and 11 IUGR (classified by umbilical artery doppler pulsatility index) at elective cesarean section, and also in 13 controls during pregnancy at a gestational age comparable to IUGR. CYP19A1 and ESRRG expressions were analyzed in placental tissue. Maternal/fetal characteristics, placental and molecular data were compared among study groups and tested for correlations. Results: Maternal E2 plasma concentrations were significantly decreased in IUGR compared to controls at delivery. When analyzing normal pregnancies at a gestational age similar to IUGR, E2 levels were not different to pathological cases. However, E2 levels at delivery positively correlated with placental efficiency. Placental CYP19A1 levels were significantly higher in IUGR placental tissue vs. controls, and specifically increased in female IUGR placentas. ESRRG expression was not different among groups. Discussion: We report a positive correlation between 17-ß Estradiol levels and placental efficiency, that might indicate a disrupted steroidogenesis in IUGR pregnancies. Moreover, we show alterations of CYP19A1 expression in IUGR placentas, possibly indicating a compensatory effect to the adverse IUGR intrauterine environment, also depending on fetal sex. Further studies are needed to deeper investigate IUGR alterations in the complex interaction among molecules involved in placental steroidogenesis.

Decreased levels of H3K9ac and H3K27ac in the promotor region of ovarian P450 aromatase mediated low estradiol synthesis in female offspring rats induced by prenatal nicotine exposure as well as in human granulosa cells after nicotine treatment.

Prenatal nicotine exposure (PNE) could induce ovarian dysplasia in offspring. This study aimed to confirm its intrauterine origin and explore a programming mechanism of ovarian dysplasia caused by PNE. Pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg.d) from gestation day (GD) 9 to GD20. Serum of female offspring was obtained for hormone assays and ovarian tissues were collected. The results showed that PNE impaired ovarian development, and inhibited estradiol production and cytochrome P450 aromatase (P450arom) expression before and after birth. Moreover, the nicotinic acetylcholine receptors (nAChRs) expression was increased in utero, while histone 3 lysine 9 acetylation (H3K9ac) and H3K27ac levels in the P450arom promoter region were decreased persistently in PNE group before and after birth. In vitro, nicotine decreased P450arom expression and estradiol production in human granulosa cell line KGN. Furthermore, nicotine treatment up-regulated nAChRα6 and α9 expression and down-regulated the H3K9ac and H3K27ac levels of the P450arom promoter region. Non-specific nAChRs inhibitor vecuronium bromide reversed these effects. These results suggest that PNE could induce ovarian dysplasia and inhibit estradiol synthesis in the female offspring rats, which was related to the decreased H3K9ac and H3K27ac levels in the promotor region of the P450arom via the nAChRs.

Aromatase immunolocalization and activity in the lizard's brain: Dynamic changes during the reproductive cycle.

The purpose of the present study is to highlight the role of aromatase in the neuroendocrine control of the reproductive cycle of the male lizard Podarcis sicula during the three significant phases, i.e. the pre-reproductive, reproductive, and post-reproductive stages. Using immunohistochemical, biochemical, and hormonal tools, we have determined the localization and the activity of P450 aromatase (P450 aro) in the lizard's brain together with the determination of hormonal profile of sex steroids, i.e. testosterone and 17ß-estradiol. The present data demonstrated that the localization of P450 is shown in brain regions involved in the regulation of the reproductive behavior (medial septum, preoptic area, and hypothalamus). Its activity, as well as the intensity of the signal, is modified according to the period of reproduction, resulting in functional dynamic changes. P450 aro activity and signal intensity decrease in the pre-reproductive period and progressively increase during the reproductive stage until it reaches the maximum peak level at the post-reproductive phase. P450 aro determines a local estrogen synthesis, balancing the testosterone and estradiol levels, and therefore its role is crucial, since it plays an important role in the neuroendocrine/behavioral regulation of the reproductive processes in the male lizard P. sicula.

Influence of various intensities of 528 Hz sound-wave in production of testosterone in rat's brain and analysis of behavioral changes.

Testosterone is a nuclear androgen receptor ligand that controls multiple pathways in brain. In addition to the active biosynthesis of steroids in classic steroidogenic organs such as gonads, adrenals and placenta, testosterone also produced in astrocyte cells of brain. Testosterone and its level must be regulated in brain; because, it directly and indirectly affects memory and several key behavioral characteristics. The significance of sound waves on key enzymes that regulate levels of testosterone in brain has not been investigated. The aim of our study was to examine physical stress of such as sound on induction behavioral changes in animal models. According to the current study, sound waves with 528 Hz frequency in 100 dB intensity induce testosterone production in brain by enhancing StAR and SF-1 and reducing P450 aromatase gene expression. Frequency of 528 Hz also reduces total concentration of reactive oxidative species in brain tissue. Prolonged exposure to this sound wave showed reduction of anxiety related behaviors in rats. The results reveal that reduced anxiety is related to increased concentration of testosterone in brain. This study may lead to ascertain a possible therapy in which sounds may be utilized to reduce anxiety in individual.

StAR protein and steroidogenic enzyme expressions in the rat Harderian gland.

The Harderian gland (HG) of the rat (Rattus norvegicus) secretes copious amounts of lipids, such as cholesterol. Here we report a study of the expressions of the StAR protein and key steroidogenic enzymes in the HG of male and female rats. The objective of the present investigation was to ascertain (a) whether the rat HG is involved in steroid production starting with cholesterol, and (b) whether the pattern of gene and protein expressions together with the enzymatic activities display sexual dimorphism. The results demonstrate, for the first time, the expression of StAR gene and protein, and Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Srd5a2 and Cyp19a1 genes in the rat HG. StAR mRNA and protein expressions were much greater in males than in females. Immunohistochemical analysis demonstrated a non-homogeneous StAR distribution among glandular cells. Hsd17b3 and Cyp19a1 mRNA levels were higher in males than in females, whereas Srd5a1 mRNA levels were higher in females than in males. No significant differences were observed in mRNA levels of Cyp11a1, Hsd3b1 and Srd5a2 between sexes. Furthermore, the in vitro experiments demonstrated a higher 5α-reductase activity in the female as compared to the male HG vice versa a higher P450 aro activity in males as compared to females. These results suggest that the Harderian gland can be classified as a steroidogenic tissue because it synthesizes cholesterol, expresses StAR and steroidogenic enzymes involved in both androgen and estrogen synthesis. The dimorphic expression and activity of the steroidogenic enzymes may suggest sex-specific hormonal effects into the HG physiology.

Synthesis of steroid hormones in the porcine oviduct during early pregnancy.

Past studies of the oviducts have documented oviductal steroid production during the oestrous cycle in pigs. The present study examined whether the pig oviducts are the source of steroid hormones during early pregnancy. In the ampulla and isthmus, the expression of 3ß-hydroxysteroid dehydrogenase (3ßHSD) and aromatase cytochrome P450 (CYP19) mRNA by real-time PCR, cellular localization and quantities of the studied proteins by immunofluorescence and Western blot analysis, and concentration of steroid hormones in oviductal flushings by radioimmunoassay, were studied. The expression of 3ßHSD in the ampulla and isthmus was correlated (r = 0.89) and higher on Days 2-3 and 15-16 than on Days 10-11 and 12-13. CYP19 expression was elevated in the ampulla on Days 2-3, 10-11 and 15-16 and in the isthmus on Days 2-3 vs. the other days studied. The studied proteins were localized in oviductal epithelial cells. In the ampulla, the quantity of 3ßHSD protein did not change, and was greater in the isthmus on Days 2-3 vs. Days 12-13 of pregnancy. The P450arom protein quantity increased in the ampulla on Days 2-3 vs. Days 10-11 and 15-16 and vs. Days 10-11 and 12-13 in the isthmus. The concentrations of progesterone and androstenedione in oviductal flushings were lowest on Days 12-13 and on Days 2-3 and 15-16, respectively, while oestradiol-17ß and oestrone levels did not change. Porcine oviducts are the sources of steroid hormones during early pregnancy. The expression of steroidogenic enzymes primarily increases during the embryos presence in the oviduct, i.e., on Days 2-3 of pregnancy.

Extremely low-frequency electromagnetic field (EMF) generates alterations in the synthesis and secretion of oestradiol-17ß (E2) in uterine tissues: An in vitro study.

An electromagnetic field (EMF) of extremely low frequency may affect physiological processes in mammals. The aim of the present study was to determine the effect of an EMF on the synthesis and secretion of oestradiol-17ß (E2) in the porcine uterus. Endometrial and myometrial slices were harvested on days 12-13 of the oestrous cycle and exposed in vitro to an EMF (50 and 120 Hz, 8 mT) for 2 and 4 h in the presence or absence of progesterone (P4). Subsequently, the incubation media were used to determine the concentration of E2 with RIA. Tissues fragments were used to study the expression of CYP19A3 mRNA using Real-Time PCR and the abundance of P450 aromatase using Western Blotting. The 50-Hz EMF increased E2 release from the endometrium and the myometrium at both time points of in vitro incubation. A 120-Hz EMF decreased the endometrial secretion of E2 after 2 h of incubation and did not affect E2 secretion after 4 h. In the myometrium, the 120-Hz EMF increased E2 secretion after 4 h of incubation. In P4-treated uterine fragments, no significant EMF exposition-related changes were observed. Only myometrial fragments incubated in the presence of P4 at 120-Hz EMF (4 h) released higher amounts of E2 due to EMF treatment. The 50-Hz EMF exposure did not change the CYP19A3 mRNA expression in endometrial fragments incubated in the presence or absence of P4. In myometrial fragments, the highest CYP19A3 mRNA expression was observed in fragments not exposed to the 50-Hz EMF and P4-treated tissues compared to that in fragments exposed to 50 Hz EMF and incubated with or without P4 and control (no EMF and no P4) fragments. The EMF at 120 Hz decreased basal endometrial CYP19A3 mRNA expression and did not change the expression in the P4-treated endometrium. In the myometrium, the EMF at 120 Hz increased CYP19A3 mRNA expression in slices incubated without P4 and had no effect in the presence of P4. The EMF exposure (50 and 120 Hz) did not affect P450 aromatase abundance in either the endometrium or the myometrium. In conclusion, the EMF induces changes in the synthesis and release of E2 in uterine tissues harvested during days 12-13 of the oestrous cycle. These changes are related to the EMF frequency used, the time of the exposition and the presence of P4. We suspect that this observed phenomenon might lead to changes in the intrauterine milieu of oestrogen, which is crucial for the proper activity of uterine tissues during the mid-luteal phase of the oestrous cycle.