Plasma E2 Levels Research Articles

Plasma Levels of Estradiol and Progesterone are Positively Associated with IL-1α and IL-1Ra in Young Women with PTSD

Introduction: Post-traumatic stress disorder (PTSD) is independently associated with greater cardiovascular disease (CVD) risk. Prior studies have reported higher inflammation as a possible underlying mechanism. Premenopausal females are typically thought to be protected from CVD due to estradiol (E2) and available data supports growing CVD risk in females before menopause by virtue of having PTSD. Given that E2 and progesterone (P4) have been shown to have a broad range of both pro- and anti-inflammatory effects, the purpose of our study was to investigate whether plasma levels of E2 and P4 are associated with subclinical inflammation in young women with PTSD. We hypothesized that E2 and P4 will be negatively associated with IL-1α, IL-1Ra, and IL-6. Methods: Seventy-five otherwise healthy young women (age, 28±7 years; BMI, 26.7±6 kg/m2) with a prior clinical diagnosis of PTSD (PTSD+, n=39) and without a PTSD diagnosis (PTSD-, n=36) were included in this study. We collected blood plasma samples to quantify E2 and P4 and inflammatory biomarkers Interleukin-1α (IL-1α), Interleukin-1 receptor antagonist (IL-1Ra) and Interleukin-6 (IL-6). Total plasma levels of E2 and P4 were measured using the quantitative sandwich enzyme immunoassay technique (ELISA). The inflammatory biomarkers were quantified using a Luminex assay. Participants were also asked to complete a self-administered PTSD checklist for DSM-5 (PCL5) to assess the severity of PTSD symptoms. We ran Pearson correlations between the sex hormones and the inflammatory biomarkers stratified by PTSD diagnosis. Results: We found a positive correlation between E2 levels and IL-1α (r=0.39, p=0.018), E2 and IL-1Ra (r=0.35, p=0.030), P4 and IL-1α (r=0.45, p=0.005), and P4 and IL-1Ra (r=0.27, p=0.101) in the PTSD+ group. In contrast, in the PTSD- group, there was no correlation between E2 and IL-1α (r=0.05, p=0.77), E2 and IL-1Ra (r=-0.06, p=0.73), P4 and IL-1α (r=-0.04, p=0.83) and P4 and IL-1Ra (r=0.07, p=0.68). Conclusion: Our analysis revealed that in young women with a clinical diagnosis of PTSD, E2 and P4 levels in the blood are positively associated with an increased IL-1α and IL-1Ra but not in young trauma-exposed women without PTSD. These findings suggest that in young women with PTSD, E2 and P4 might have a paradoxical effect on inflammation such that E2 and P4 promote the release of both pro-inflammatory (i.e. IL-1α) and anti-inflammatory (i.e. IL-1Ra) cytokines. Conversely, an increase in subclinical inflammation could lead to an increase in E2 and P4 levels, which in turn would promote the release of anti-inflammatory cytokines in order to inhibit the inflammatory response. K01HL161027 and UMN CTSI UL1TR002494 No Disclosure. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

17 β-Estradiol ameliorates cardiac parasympathetic dysfunction following myocardial infarction in male mice

Background: Vagal dysfunction after chronic myocardial infarction (MI) is associated with an increased risk of ventricular tachycardia (VT)/ventricular fibrillation (VF) and increased mortality in heart failure. The incidence and outcomes of VT/VF are influenced by sex, with women having a lower incidence of VT/VF associated with ischemic heart disease. However, the mechanisms behind these sex differences remain unclear and could be due to sex differences in autonomic remodeling post-MI that is mediated by estrogen. Hypotheses: We hypothesized that estrogen improves vagal function after MI by reducing oxidative stress in vagal sensory neurons and improving vagal afferent/efferent reflexes and function. Methods: Mice with expression of channelrhodopsin (ChR2) in specific sensory afferent neurons were created by crossing vesicular glutamate transporter 2 (VGlut2)-IRES-Cre mice with ChR2-EYFP mice, and their offspring (Vglut2-ChR2-EYFP) used for optogenetic stimulation studies. Sham surgery (n=6) or MI (n=6) was performed in two separate groups of male mice. A third group of male mice (n=8) underwent 17β-Estradiol (E2) pellet (0.5 milligrams/pellet) implantation followed by MI (E2+MI) after 2.5 weeks. Terminal studies were performed 2-3 weeks post-MI/sham procedures by placing a blue light (473 nm) laser probe over the left cervical vagus in vivo to stimulate vagal sensory neurons (20 Hz, 10 & 20 msec stimulations for 5 sec). Heart rate (HR) and blood pressure (BP) responses were measured in response to optogenetic stimulation. Plasma E2 and nodose ganglion 4-hydroxynaneol (4HNE) levels, a marker of lipid peroxidation, were evaluated. Results: In response to optogenetic vagal stimulation, MI had reduced reflex heart rate responses vs. sham animals, while E2+MI mice demonstrated significantly better responses vs. MI and similar responses to sham animals (20 Hz, 10 msec: sham -59 ± 3%, MI -24 ± 3%, E2+MI -58 ± 4%, MI vs. E2+MI, P < 0.001; 20 Hz, 20 msec: sham -59 ± 4%, MI -31 ± 3%, E2+MI -60 ± 3%, MI vs. E2+MI, P < 0.001). In addition, while BP responses post-MI were reduced vs. sham, E2+MI male mice also demonstrated greater BP responses to optogenetic stimulation (20 Hz, 10 msec: sham -28 ± 4%, MI -12 ± 1%, E2+MI -27± 1%, MI vs. E2+MI, P < 0.001; 20 Hz, 20 msec: sham -32 ± 4%, MI -13 ± 1%, E2+MI -27 ± 5%, MI vs. E2+MI, P < 0.001). 4HNE levels were increased in MI vs. sham animals, while E2+MI mice demonstrated significant reductions in 4HNE levels (Sham 0.17 ± 0.09 μg/protein, MI 0.77 ± 0.11 μg/protein, E2±MI 0.26 ± 0.06 μg/protein, MI vs. E2+MI, P < 0.001). Finally, plasma measurements confirmed higher levels of estrogen in E2-implanted mice (MI 48 ± 10 pg/mL, E2+MI 850 ± 170 pg/mL, MI vs. E2+MI, P < 0.001). Conclusions: MI causes pathological vagal remodeling that is reflected in decreased sensory vagal neurotransmission and increased oxidative stress in the vagal ganglia. Parasympathetic dysfunction and oxidative stress are mitigated by estrogen, suggesting that estrogen may be an important factor in mitigating the resulting pathological autonomic remodeling post-MI that predisposes to VT/VF. NIHR01HL148190. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

(007) Impact of Non-Invasive Oestrus Synchronization on Sexual Behavior in Intact Sprague Dawley Rats

Abstract Introduction Female sexual behavior animal translation data do not translate to acute results and neither current synchronization invasive models (ovariectomy+Estrogen “E2”+Progesterone “P4”) guarantee hormonal physiological values. Objective Therefore, our study investigated the effect of non-invasive oestrus synchronization on sexual behavior. Methods Methods: Twelve week old Sprague Dawley intact rats were synchronized and randomly divided into three groups each of n=8-10. Each group received a dose of prostaglandin F2α “PGF2α” (75, 125 and 250 μg) at days 0 and 3, and 1 mg of progesterone at day four. Vaginal cytology was performed to determine the point of the cycle and the animals underwent an incentive test. To investigate the sexual behavior, the synchronized female rats were paired with trained male rats, and behavior recorded. The videos were quantified by trained operators. Sexual behavior was quantified as paracopulatory (hops, darts, ear wiggling, sniffing from male and from female) or copulatory (male mounting female, thrusting from male, intromission, ejaculation, female for sniffing position, lordosis, orgasm, female mounting on male, female rotation in her axis and female licking itself), E2 and Follicle Stimulating hormone (FSH) in plasma and E2 in vaginal fluid were quantified by immune assay. Results Based on the vaginal cytology, the proportion of animals in proestrus (p), estrus (e) or metestrus (m) was calculated with values of 0.5 (p), 0.25 (e) and 0.25 (m) for the group which received 0.75 μg PGF2α; 0.4 (p), and 0.6 (e) for the group which received 0.125 μg PGF2α; 0.2 (p), 0.5 (e), and 0.3 (m) for the group which received 250 μg PGF2α. The numbers of hops was higher in the proestrus compared to the metestrus, and of mounts higher in the estrus compared to the metestrus phase. The sexual encounter profile and E2 levels were unchanged with the different PGF2α doses, but in the high-dose group no hops were observed. The levels of sexual behavior was comparable to previous studies of hormone substituted ovariectomized rats, but E2 and FSH plasma levels were in a physiological range. Conclusions In summary, the non-invasive synchronization protocol could substitute the state-of-the-art used ovariectomized+hormone (E2) model. Our findings suggest the largest part of rats were synchronized in the oestrus phase with 125 μg PGF2α. but the 250 μg had also a positive profile. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Initiator Pharma A/S.

Intraperitoneal administration of kisspeptin-10 modulates follicle maturation, gonadal steroids, calcium and metabolites in Sterlet sturgeon, Acipenser ruthenus

Kisspeptin is a multifunctional neurohormone, primarily involved in the regulation of reproduction. We tested whether peripheral administration of kisspeptin10 (KP-10) via intraperitoneal injection or slow release affects reproductive hormones and metabolites in Sterlet sturgeon (Acipenser ruthenus). Plasma and mucus 17β-estradiol (E2), and testosterone (T), plasma and follicular vitellogenin (VTG) and calcium (Ca) as well as glucose and lipids were determined. Mature Sterlet sturgeon were grouped into six groups: saline i.p injection (control), human kisspeptin (hKP-10) i.p injection; acipenser kisspeptin (aKP-10) i.p injection; hKP-10 (slow release); aKP-10 (slow-release) and no treatment control. No effect for KP-10 on sturgeon body weight was found after 4 weeks of treatment. Multivariate analysis revealed a significant disparity in plasma E2 levels. It was significantly different between groups (time, P = 0.0022). E2 in epithelia mucosa showed significant difference between and within groups in the acute group (time, P = 0.0252; treatment, P = 0.0423; time × treatment, P = 0.0429). T levels were unaffected by treatments (P > 0.05). The presence of synthetic aKP-10 led to an elevation in oocyte and plasma VTG levels (P < 0.05). Prolonged exposure to this peptide resulted in an increase in plasma calcium levels. Simultaneously, there was an augmentation in the number of mature follicles. Regardless of the duration of exposure, aKP-10 significantly elevated plasma glucose levels in Sterlet (P < 0.0). Additionally, KP-10 led to an increase in plasma lipids and cholesterol in Sterlet. Overall, our data support an involvement for KP-10 in the regulation of gonadal steroid hormones, oocyte maturation and metabolite levels in sturgeon, suggesting a positive role for this peptide in the reproductive physiology of this species.

High mobility group box protein 1 sensitizes mononuclear cells to further contact with lipopolysaccharide.

Fever is an adaptive host-defense response to infection and nowadays is rightly considered to be an expression of a healthy body and a well-functioning immune system. The condition is that it must be tightly regulated. Therefore, in individual cases, fever may be detrimental and should be treated. Specific excessive febrile reaction to pathogens which occurs after aseptic injuries is one among such cases. We previously found that among necrotic products, high mobility group box protein 1 (HMGB1) released from the site of aseptic injury affects immune effectors (cells) to mediate higher fever in response to further contact with bacterial lipopolysaccharide (LPS). Here we observed that intraperitoneal (i.p.) pre-injection of recombinant HMGB1 (5 µg/rat i.p.) provoked an increase in plasma levels of prostaglandin E2 (PGE2) in rats and augmented release of interleukin (IL)-1β and IL-6 after LPS administration at a dose of 50 µg/kg i.p. compared to rats pre-injected with saline or heat-denatured HMGB1. Furthermore, peripheral blood mononuclear cells (PBMCs) isolated from rats injected with HMGB1 were more sensitized to produce enhanced levels of IL-1β and PGE2 when stimulated with LPS in vitro (1 µg/ml/106 cells for 4 h) compared to control animals injected with saline or heat-denatured HMGB1. We also noted a significant increase in activation of nuclear factor κB (NF-κB) in cells isolated from rats injected with HMGB1. Altogether, the obtained results suggest that HMGB1 participates in priming of immune cells to further contact with pathogens.

Steroid profile and ovarian gene expression in Genyatremus luteus (Teleostei, Perciformes: Haemulidae) during sexual maturation

The aim of the present study was to assess histological features, sex steroid profile, and ovarian gene expression throughout the ovarian maturation of Genyatremus luteus in Brazilian Coastal Equatorial Amazon. Specimens were categorized into different gonadal maturation stages–immature (n = 7), maturing (n = 7), mature (n = 7), and spawned (n = 7)–based on histological features of ovarian follicles and on oocyte development. Plasma E2, 11-KT and 17α-OHP levels were measured through enzyme-linked immunosorbent assays. The gene expression of estrogen (er) and LH (lhr) receptors in the ovary was analyzed through real-time polymerase chain reaction. The highest plasma E2 concentration was found in mature specimens (P&lt; 0.05). Plasma 17α-OHP concentrations have significantly increased from the immature to the final maturation stage (P&lt; 0.05). Maturational reproductive stage did not have any effect on plasma 11-KT concentrations. Er expression in ovaries has significantly increased throughout ovarian maturation (P &lt; 0.05), and it remained high at mature and spawned stages. Lhr expression showed similar trend throughout the maturation process, although its peak was observed at spawned stage (P&lt; 0.05). In conclusion, the gene expression profile of er and lhr and plasma E2 and 17α-OHP concentrations are proportional to oocyte development; they play important role in regulating ovarian development and ovulation in G. luteus.

SAT434 Estradiol Controls GnRH-II Gene Expression In The Hypothalamus And Pituitary Gland Of Female Rhesus Macaques (Macaca Mulatta)

Abstract Disclosure: A. Lomniczi: None. M. Appleman: None. H.F. Urbanski: None. It has been known for many years that rhesus macaques express two forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) in the medial basal hypothalamus (MBH), and that both forms are capable of stimulating the release of luteinizing hormone (LH) in vivo. However, while much has been published about the role of GnRH-I in reproduction, very little is known about the function of GnRH-II. To shed light on this issue, we studied the expression pattern of these two genes in different parts of the monkey hypothalamus, median eminence and pituitary gland. Quantitative PCR (qPCR) analysis revealed that while GnRH-I is preferentially expressed in the Arcuate Nucleus (ARC), Median Eminence (ME), Supraoptic Nucleus (SON) and Suprachiasmatic Nucleus (SCN); GnRH-II was highly expressed in the SON, ARC-ME and Paraventricular Nucleus (PVN), confirming our previously published in situ hybridization studies. Strikingly, GnRH-II mRNA was also detected in the pituitary gland, a site where GnRH-I was not found. We have previously demonstrated that maximum hypothalamic GnRH-II expression occurs around the time of the preovulatory LH surge, when estrogen (E2) levels are maximal, but no significant changes in GnRH-I mRNA levels occur. Here using an ovariectomized (OVX) macaque model subjected to E2 replacement paradigm, designed to mimic the plasma E2 levels observed during the preovulatory estradiol peak, we performed RNAseq and qPCR from the PVN and pituitary gland to identify genes differentially regulated by E2. While GnRH-I mRNA was not affected by E2 treatment in any of the tissues studied, pituitary GnRH-II expression highly correlated with estrogen levels (r&amp;gt;0.8; p&amp;lt;0.01), with over a hundred-fold increase in mRNA expression. Taken together, these data suggest that pituitary GnRH-II (but not GnRH-I) gene expression is induced by a positive feedback action of estradiol. This novel finding raises the possibility that GnRH-II plays a major role in triggering the preovulatory LH surge in primates, not only at the ARC-ME but also at the pituitary level. Presentation Date: Saturday, June 17, 2023

Decamethylcyclopentasiloxane affects estradiol production in female rats but not H295R cells.

Sex hormones, such as androgens and estrogens, are predominantly produced in the gonads (ovaries and testes) and adrenal cortex. Endocrine-disrupting chemicals (EDCs) are substances that mimic, block, or interfere with hormones in the endocrine systems of humans and organisms. EDCs mainly act via nuclear receptors and steroidogenesis-related enzymes. In the OECD conceptual framework for testing and assessment of EDCs, several well-known assays are used to identify the potential disruption of nuclear receptors both in vivo and in vitro, whereas the H295R steroidogenesis assay is the only assay that detects the disruption of steroidogenesis. Forskolin and prochloraz are often used as positive controls in the H295R steroidogenesis assay. Decamethylcyclopentasiloxane (D5) was suspected one of EDCs, but the effects of D5 on steroidogenesis remain unclear. To establish a short-term in vivo screening method that detects the disruption of steroidogenesis, rats in the present study were fed a diet containing forskolin, prochloraz, or D5 for 14 days. Forskolin increased plasma levels of 17β-estradiol (E2) and testosterone as well as the mRNA level of Cyp19 in both the adrenal glands and ovaries. Prochloraz induced the loss of cyclicity in the sexual cycle and decreased plasma levels of E2 and testosterone. D5 increased E2 levels and shortened the estrous cycle in a dose-dependent manner; however, potential endocrine disruption was not detected in the H295R steroidogenesis assay. These results demonstrate the importance of comprehensively assessing the endocrine-disrupting effects of chemicals on steroidogenesis in vivo.

Embryonic benzophenone-3 exposure inhibited fertility in later-life female zebrafish and altered developmental morphology in offspring embryos.

Benzophenone-3 (BP3), an organic UV filter widely used in personal care products, is ubiquitous in aquatic environments. Previous studies have shown that BP3 can interfere with oocytes development in the ovary. The current study was conducted to evaluate the effects of embryonic BP3 exposure on reproductive outcomes in later life. Zebrafish embryos were exposed to different concentrations of BP3 (0, 1, 10, 100 μg/L) for 5 days in the developmental stage and subsequently fed for 4 months without any toxins. The body length, body weight, and ovary weight in F0 female adult zebrafish and morphology indices in F1 offspring embryos were measured. The reproductive behaviors of adult zebrafish were recorded by a digital camera. HE staining was used to estimate the development of oocytes and the proportion of different phases was calculated. qPCR was used to detect the expression levels of reproduction-related genes of the hypothalamic-pituitary-gonadal (HPG) axis. Our findings revealed that the body length and body weight were not changed with embryonic BP3 exposure, but BP3 exposure inhibited the development and maturation of ovaries in later-life female zebrafish, accompanied by an increased proportion of follicles in the primary growth and early vitellogenic stages, and a decline in the full-growth stage in ovaries. Meanwhile, reduced egg production, delayed hatching rate, altered somite count and increased mortality rate were observed at 100 μg/L in offspring embryos. Behavioral results showed that BP3 exposure reduced the frequency of chasing, touching, entering the spawning area, and the duration of fish entering the spawning area later in life, qPCR analysis showed that the expression levels of reproduction-related genes of the HPG axis were downregulated in females, following a decreasing trend in plasma E2 and 11-KT levels. These results suggested that embryonic BP3 exposure negatively affected the fertility of fish and the development of their offspring embryos, which may cause potential risks to aquatic ecosystems and human health.

YB-1 Protein Prevents Age Decline in Plasma Estradiol in 5xFAD Transgenic Aging Female Mice

Alzheimer’s disease (AD) is an incurable neurodegenerative disease that is the main cause of dementia in the elderly. When looking for new treatments for AD, attention was drawn to the multifunctional Y-box-binding protein 1 (YB-1). Previously, we revealed a positive effect of intranasal administration of YB-1 on learning and spatial memory, along with a decrease in the content of cerebral β-amyloid and the intensity of plaque initiation, with an improvement in the survival of neurons in the cortex and hippocampus of male AD mice. However, AD affects women twice as often as men, so it is of great interest to study the effects of YB-1 on aging females. Estrogens and androgens are necessary for the maintenance of cognitive function during aging and, apparently, may prevent the development of AD. In this work, peripheral levels of estradiol (E2) and cytokines were studied after intranasal administration of YB-1 to aging female 5xFAD transgenic mice and control non-transgenic animals. In intact aging animals of both groups, a violation of the estrous cycle and a decrease in the level of E2 in blood plasma were revealed. Mice treated with YB-1 did not show a characteristic age-related decrease in plasma E2 levels. The introduction of YB-1 did not affect the peripheral level of cytokines. Thus, a novel, previously undescribed effect of YB-1 on plasma E2 levels in aging female mice is shown. These data indicate that YB-1 may be a promising compound in the prevention and treatment of neurodegenerative diseases. However, further experiments are needed to gain insight into the detailed mechanisms of YB-1 action.

Evaluation of 28-day estradiol and progesterone vaginal rings in a phase 1 clinical pharmacokinetic study.

The aim of this work is to develop a combination of 17β-estradiol (E2) and progesterone (P4) in a single-dose intravaginal ring (IVR) for the treatment of vasomotor symptoms (VMS) and genitourinary syndrome of menopause while providing endometrial protection. The objective of this study was to evaluate DARE-HRT1, a 28-day IVR that continuously delivers E2 and P4, in a phase 1 clinical trial to assess its pharmacokinetics. This was an open-label, three-arm (group) study. Thirty-two (32) healthy postmenopausal women were recruited at two Australian sites. The average age was 57.2 years (47-69 y). The first arm received one ring for 28 days designed to release E2 at a rate of 80 μg/d and P4 at 4 mg/d (80/4 IVR); the second arm received a ring releasing E2 at 160 μg/d and P4 at 8 mg/d (160/8 IVR). The third arm received oral Estrofem (1 mg E2) and Prometrium (100 mg P4) both daily for 29 days. Blood samples were taken predose then intensively over the first day (day 1) and periodically thereafter over the remaining 27 days. After removal of the rings on the morning of day 29, intensive samples were collected. Similar procedures were conducted with women enrolled in the oral group. The plasma samples were analyzed for E2, estrone (E1), and P4 using validated bioanalytical methods. The baseline-adjusted steady-state plasma levels of E2 and P4 from 80/4 IVR were 20.4 ± 17.1 pg/mL and 1.32 ± 0.19 ng/mL (n = 10), respectively. The baseline-adjusted steady-state plasma levels of E2 and P4 from 160/8 IVR were 30.9 ± 8.7 pg/mL and 2.08 ± 0.50 ng/mL (n = 10), respectively. The baseline-adjusted average plasma concentrations of E2 and P4 at day 29 of the oral group were 35.4 ± 11.2 pg/mL and 0.79 ± 0.72 ng/mL (n = 11), respectively. The baseline-adjusted steady state of E1 from the 80/4 IVR and the 160/8 IVR were 22.1 ± 16.6 pg/mL (n = 10) and 25.2 ± 12.3 pg/mL (n = 10), respectively. The baseline-adjusted concentration of E1 in the oral arm was 209 ± 67.7 ng/mL (n = 11). The IVR were well tolerated, and no serious adverse events were reported. The 80/4 IVR and 160/8 IVR gave similar steady-state concentrations of E2 as seen with drug products approved by the US Food and Drug Administration for treatment of VMS and genitourinary symptoms of menopause. The E2 concentrations of this study support the potential of DARE-HRT1, a promising new option for hormone therapy for treatment of VMS and vaginal symptoms associated with menopause.

Transcriptomic analysis reveals the molecular mechanisms underlying osteoclast differentiation in the estrogen-deficient pullets

Several previous reports have suggested that estrogen (E2) is a vital signal responsible for the regulation of skeletal homeostasis and bone remodeling in mammals. E2 could efficiently accelerate the growth of medullary bone in pullets during sexual maturity. Furthermore, the low E2 level can strengthen the mechanical bone functions in female hens. However, mechanistic studies to describe the effects of E2 on bone in pullets during the initiation of the puberty period are remaining elusive. Therefore, the aim of this study was to explore the effect of inhibiting E2 biosynthesis on the biomechanical properties and its molecular mechanism during sexual maturity of pullets. In this study, a total of 90 Hy-line Sonia pullets with comparable body weight at 13 wk of age were selected and categorized into 2 separate groups. Daily, 0.5 mg/4 mL of letrozole (LZ) was orally administered to the treatment (TRT) group and 4 mL of saline to the control (CON) group of pullets for 6 wk. Compared with the CON group, a lower plasma E2 level was observed in the TRT group. Furthermore, plasma P, Gla protein (BGP), and 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3) levels were markedly suppressed, whereas the plasma alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) levels were significantly elevated. Moreover, the cortical bone thickness and breaking strength of the tibia and femur, the bone mineral density of the humerus, and the bone mineral content of the humerus as well as the femur were increased significantly. The expression levels of 340 differentially expressed genes (DEGs) differed significantly between the CON and TRT group in the tibia at 19 wk of age. Among them, 32 genes were up-regulated, whereas 308 were down-regulated in the TRT group. The variations in candidate genes associated with osteoclast differentiation and cell adhesion may indicate that LZ inhibits E2 biosynthesis, consequently, reduces osteoclast differentiation by suppressing inter-cellular communication and cells attaching to extracellular matrix components. Taken together, the present study demonstrated that inhibiting E2 synthesis during sexual maturity of pullets decreased osteoclast differentiation and considerably enhanced bone quality.

Combined Treatment with KV Channel Inhibitor 4-Aminopyridine and either γ-Cystathionine Lyase Inhibitor β-Cyanoalanine or Epinephrine Restores Blood Pressure, and Improves Survival in the Wistar Rat Model of Anaphylactic Shock.

Simple SummaryAllergic diseases are presenting a constant increase all over the world and caused by such different substances as food, drugs, and pollens. Anaphylactic shock is the more severe complication of allergy which can induce death if the treatment is not administered immediately. Some patients do not respond to the recommended treatment, intra venous or intramuscular epinephrine. The pathophysiology of anaphylactic shock is still under investigation. The mediators released after the activation of mast cells and basophiles act on endothelial cells and smooth muscle cells, inducing the vasodilation responsible for hypotension and shock. Nitric oxide and hydrogen sulphide are both intracellular mediators that induce vasodilation. The role of potassium voltage dependent channels is suspected. We aimed to demonstrate the ability of a blocker of potassium voltage dependent channels, 4-aminopyridine, alone or in combination with inhibitors of cystathionine γ-lyase to restore blood pressure and improve survival in an ovalbumin rat anaphylactic shock model. The blockade of potassium voltage dependent channels alone or combined with inhibitors of cystathionine γ-lyase, dl-propargylglycine, or β-cyanoalanine restored blood pressure and improved survival. These findings suggest possible investigative treatment pathways for research concerning epinephrine-refractory anaphylactic shock in patients.The mechanism of anaphylactic shock (AS) remains incompletely understood. The potassium channel blocker 4-aminopyridine (4-AP), the inhibitors of cystathionine γ-lyase (ICSE), dl-propargylglycine (DPG) or β-cyanoalanine (BCA), and the nitric oxide (NO) synthase produce vasoconstriction and could be an alternative for the treatment of AS. The aim of this study was to demonstrate the ability of L-NAME, ICSE alone or in combination with 4-AP to restore blood pressure (BP) and improve survival in ovalbumin (OVA) rats AS. Experimental groups included non-sensitized Wistar rats (n = 6); AS (n = 6); AS (n = 10 per group) treated i.v. with 4-AP (AS+4-AP), epinephrine (AS+EPI), AS+DPG, AS+BCA, or with L-NAME (AS+L-NAME); or AS treated with drug combinations 4-AP+DPG, 4-AP+BCA, 4-AP+L-NAME, or 4-AP+EPI. AS was induced by i.v. OVA (1 mg). Treatments were administered i.v. one minute after AS induction. Mean arterial BP (MAP), heart rate (HR), and survival were monitored for 60 min. Plasma levels of histamine, prostaglandin E2 (PGE2) and F2 (PGF2α), leukotriene B4 and C4, angiotensin II, vasopressin, oxidative stress markers, pH, HCO3, PaO2, PaCO2, and K+ were measured. OVA induced severe hypotension and all AS rats died. Moreover, 4-AP, 4-AP+EPI, or 4-AP+BCA normalized both MAP and HR and increased survival. All sensitized rats treated with 4-AP alone or with 4-AP+BCA survived. The time-integrated MAP “area under the curve” was significantly higher after combined 4-AP treatment with ICSE. Metabolic acidosis was not rescued and NO, ICSE, and Kv inhibitors differentially alter oxidative stress and plasma levels of anaphylactic mediators. The AS-induced reduction of serum angiotensin II levels was prevented by 4-AP treatment alone or in combination with other drugs. Further, 4-AP treatment combined with EPI or with BCA also increased serum PGF2α, whereas only the 4-AP+EPI combination increased serum LTB4. Serum vasopressin and angiotensin II levels were increased by 4-AP treatment alone or in combination with other drugs. Moreover, 4-AP alone and in combination with inhibition of cystathionine γ-lyase or EPI normalizes BP, increases serum vasoconstrictor levels, and improves survival in the Wistar rat model of AS. These findings suggest possible investigative treatment pathways for research into epinephrine-refractory anaphylactic shock in patients.

Phylogenomic and expression analysis of Colossoma macropomum cyp19a1a and cyp19a1b and their non-classical role in tambaqui sex differentiation

The genes coding for Cytochrome P450 aromatase (cyp19a1a and cyp19a1b) and estrogen (E2) receptors (esr1, esr2a and esr2b) play a conserved role in ovarian differentiation and development among teleosts. Classically, the “gonad form” of aromatase, coded by the cyp19a1a, is responsible for the ovarian differentiation in genetic females via ligation and activation of the Esr, which mediates the endocrine and exocrine signaling to allow or block the establishment of the feminine phenotype. However, in neotropical species, studies on the molecular and endocrine processes involved in gonad differentiation as well as on the effects of sex modulators are recent and scarce. In this study, we combined in silico analysis, real-time quantitative PCR (qPCR) assay and quantification of E2 plasma levels of differentiating tambaqui (Colossoma macropomum) to unveil the roles of the paralogs cypa19a1a and cyp19a1b during sex differentiation. Although the synteny of each gene is very conserved among characids, the genomic environment displays striking differences in comparison to model teleost species, with many rearrangements in cyp19a1a and cyp19a1b adjacencies and transposable element traces in both regulatory regions. The high dissimilarity (DI) of SF-1 binding motifs in cyp19a1a (DI = 10.06 to 14.90 %) and cyp19a1b (DI = 8.41 to 13.50 %) regulatory region, respectively, may reflect in an alternative pathway in tambaqui. Indeed, while low transcription of cyp19a1a was detected prior to sex differentiation, the expression of cyp19a1b and esr2a presented a large variation at this phase, which could be associated with sex-specific differential expression. Histological analysis revealed that anti-estradiol treatments did not affect gonadal sex ratios, although Fadrozole (50 mg kg−1 of food) reduced E2 plasma levels (p < 0,005) as well cyp19a1a transcription; and tamoxifen (200 mg kg−1 of food) down regulated both cyp19a1a and cyp19a1b but did not influence E2 levels. Altogether, our results bring into light new insights about the evolutionary fate of cyp19a1 paralogs in neotropical fish, which may have generated uncommon roles for the gonadal and brain forms of cyp19a1 genes and the unexpected lack of effect of endocrine disruptors on tambaqui sexual differentiation.

Growth, sexual transition, and maturation of blacktip grouper Epinephelus fasciatus under long-term artificial rearing: Puberty and its associated physiological and endocrine changes

The blacktip grouper Epinephelus fasciatus is considered a valuable marine fish for aquaculture. This study was conducted to obtain information on its age of puberty, growth performance, gonadal development, sexual transition pattern, and plasma sex steroid levels from juvenile stage to maturity in specimens reared in captivity. The stages of gonadal development were identified by histological analysis, and plasma sex steroid levels were measured using an enzyme-linked immunosorbent assay. Our results show that maturation and spawning in blacktip groupers occurred at almost 3 years of age; although some females matured at 2 years. Plasma 17β-estradiol (E2) levels increased rapidly to high levels during puberty at 3 years of age, especially during the late stage of vitellogenesis at the tertiary yolk stage, and reduced at the ripe and ovulated stages prior to spawning. The gonadal somatic index also showed an increase consistent with the rise in plasma E2 levels. Plasma 11-ketotestosterone (11-KT) levels increased dramatically during sex change and in males. The significant decrease in plasma E2 levels and remarkable increase in plasma 11-KT were the key factors for the progress of sex change in these fish. Interestingly, there were the concomitant appearances of spermatocytes in ovaries of immature females at 1 year 3 months of age. The earliest appearance of sex-transitioning and male fish in blackfish groupers was observed at 2 years, although no mature females were observed below this age. This indicated that the sex change and male transformation in these fish took place without them undergoing the mature female stage. In conclusion, this species had the ability to reach puberty at 2 years of age, but most individuals matured and were functional at 3 years old. Puberty might be affected by age rather than size. In addition, although this species had a protogynous hermaphrodite pattern, when individuals born in the same year were reared together, some individuals were capable of directly becoming males without experiencing a mature female stage. This was due to the lack of socialization in captivity wherein individuals of various ages do not coexist, unlike that in the wild.

Prognostic Value of the Grade and Extent of Coronary Microvascular Dysfunction in Takotsubo Syndrome

The genes coding for Cytochrome P450 aromatase (cyp19a1a and cyp19a1b) and estrogen (E2) receptors (esr1, esr2a and esr2b) play a conserved role in ovarian differentiation and development among teleosts. Classically, the “gonad form” of aromatase, coded by the cyp19a1a, is responsible for the ovarian differentiation in genetic females via ligation and activation of the Esr, which mediates the endocrine and exocrine signaling to allow or block the establishment of the feminine phenotype. However, in neotropical species, studies on the molecular and endocrine processes involved in gonad differentiation as well as on the effects of sex modulators are recent and scarce. In this study, we combined in silico analysis, real-time quantitative PCR (qPCR) assay and quantification of E2 plasma levels of differentiating tambaqui (Colossoma macropomum) to unveil the roles of the paralogs cypa19a1a and cyp19a1b during sex differentiation. Although the synteny of each gene is very conserved among characids, the genomic environment displays striking differences in comparison to model teleost species, with many rearrangements in cyp19a1a and cyp19a1b adjacencies and transposable element traces in both regulatory regions. The high dissimilarity (DI) of SF-1 binding motifs in cyp19a1a (DI = 10.06 to 14.90 %) and cyp19a1b (DI = 8.41 to 13.50 %) regulatory region, respectively, may reflect in an alternative pathway in tambaqui. Indeed, while low transcription of cyp19a1a was detected prior to sex differentiation, the expression of cyp19a1b and esr2a presented a large variation at this phase, which could be associated with sex-specific differential expression. Histological analysis revealed that anti-estradiol treatments did not affect gonadal sex ratios, although Fadrozole (50 mg kg−1 of food) reduced E2 plasma levels (p < 0,005) as well cyp19a1a transcription; and tamoxifen (200 mg kg−1 of food) down regulated both cyp19a1a and cyp19a1b but did not influence E2 levels. Altogether, our results bring into light new insights about the evolutionary fate of cyp19a1 paralogs in neotropical fish, which may have generated uncommon roles for the gonadal and brain forms of cyp19a1 genes and the unexpected lack of effect of endocrine disruptors on tambaqui sexual differentiation.

Children with Prepubertal Anorexia Nervosa: assessing the sex hormone profile and prognostic factors for inpatient treatment outcomes.

Even though Anorexia Nervosa (AN) is an emerging research topic, scarce literature data are available on children with prepubertal AN. To date, no specific study has assessed the potential prognostic factors on treatment outcomes in this population. Observational, retrospective study of 39 children (12.5±1.8 years) hospitalized for AN, with primary amenorrhea and absence of pubertal development at clinical examination. Sexual hormonal profile: plasma levels luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), progesterone (P4), and prolactin (PRL) were assessed at admission. Clinical (age, duration of illness, comorbidity, admission body-mass index - BMI), treatment (psychopharmacological therapies) and outcome (BMI improvement, length of hospital stay - LOS) variables were assessed. Multiple linear regressions were conducted to assess potential predictors of outcomes. A predictive model of BMI improvement (F(4, 32)=8.713; Adjusted R2=0.491; p<0.001) was documented, with longer LOS (B=0.010; p<0.001), lower plasma levels of E2 (B=-0.043; p=0.002) and lower plasma levels of LH (B=-0.292, p=0.043) predicting a greater BMI improvement. A predictive model of LOS (F(2, 38)=9.527; Adjusted R2=0.310; p<0.001) was documented as well, with younger age (B=17.835; p<0.001), and reduced BMI improvement (B=22.656; p=0.012) predicting a shorter LOS. No predictive value of comorbidity, FSH, P4 or PRL was found. This is the first study to investigate the prognostic value of sex hormones on the treatment of children with prepubertal AN. Low E2, LH, and age, but not FSH, P4 or PRL, were associated with better outcomes. These data should be investigated in wider populations and controlled studies.

Ischemic preconditioning does not prevent placental dysfunction induced by fetal cardiac bypass.

Remote ischemic preconditioning (rIPC) has been applied to attenuate tissue injury. We tested the hypothesis that rIPC applied to fetal lambs undergoing cardiac bypass (CB) reduces fetal systemic inflammation and placental dysfunction. Eighteen fetal lambs were divided into three groups: sham, CB control, and CB rIPC. CB rIPC fetuses had a hindlimb tourniquet applied to occlude blood flow for four cycles of a 5-minperiod, followed by a 2-minreperfusion period. Both study groups underwent 30 min of normothermic CB. Fetal inflammatory markers, gas exchange, and placental and fetal lung morphological changes were assessed. The CB rIPC group achieved higher bypass flow rates (p < .001). After CB start, both study groups developed significant decreases in PaO2 , mixed acidosis, and increased lactate levels (p < .0004). No significant differences in tissular edema were observed on fetal lungs and placenta (p > .391). Expression of Toll-like receptor 4 and intercellular adhesion molecule-1in the placenta and fetal lungs did not differ among the three groups, as well as with vascular cell adhesion molecule-1(VCAM-1) of fetal lungs (p > .225). Placental VCAM-1 expression was lower in the rIPC group (p < .05). Fetal interleukin-1 (IL-1) and thromboxane A2 (TXA2) levels were lower at 60 min post-CB in the CB rIPC group (p < .05). There were no significant differences in tumor necrosis factor-α, prostaglandin E2, IL-6, and IL-10 plasma levels of the three groups at 60-minpost-bypass (p > .133). Although rIPC allowed increased blood flow during fetal CB and decreasedIL-1 and TXA2 levels and placental VCAM-1, it did not prevent placental dysfunction in fetal lambs undergoing CB.

Reproductive endocrine variation in female olive flounder with different stress coping styles during the breeding season

The hypothalamic–pituitary–inter-renal (HPI) axis activity is related to the stress coping styles of olive flounder, Paralichthys olivaceus. But the relationship between hypothalamic–pituitary–gonadal (HPG) axis activity and stress coping styles remains unknown. Here, we combine physiological measurements with ovarian development to examine the reproduction differences between proactive and reactive flounders. The GSI was significantly higher in the proactive fish than the reactive type during the breeding season. In April, all the proactive fish under investigation had ovaries at mid-vitellogenic stage while reactive fish had ovaries at pre-vitellogenic stage, but both the proactive fish and the reactive ones had ovaries at post-vitellogenic stage in May. In HPG axis, the plasma E2 levels and the mRNA levels of fshβ, fshr and lhcgr was higher in the proactive flounder than the reactive ones in April yet there was no difference between them in May. In HPI axis, the concentration of cortisol was lower in the proactive fish than the reactive ones in April, yet the results indicate no difference in May. Surprisingly, the transcript gr in ovary displays no differences between the proactive and the reactive fish and mr shows significant up-regulation for the proactive fish in April and the results indicated no difference again in May. Overall, these data suggested that the proactive individuals matured earlier than the reactive ones, the activity of HPI axis was lower, and the activity of HPG axis was higher in the proactive fish than the reactive ones during the breeding season.

Examining Vascular Responses to Passive Movement in Premenopausal Females: Comparisons Across Sex and Menstrual Cycle Phase

PurposeVascular responses to passive movement represent a novel assessment of vascular function and serves as a critical component for adequate blood flow regulation during exercise. Although alterations to estradiol (E2) throughout the menstrual cycle, as well as sex differences, are known to impact vascular function, limited research exists in determining whether vascular responses to passive movement are altered in premenopausal females when compared across sex or menstrual cycle phase. We hypothesized that there would be no differences in leg blood flow responses to passive leg movement (PLM) in premenopausal females when compared to age‐matched males or when evaluated across follicular menstrual cycle phases, when E2 fluctuates the most.MethodsVascular responses to one‐minute of PLM were assessed in eleven premenopausal females (n = 21; 23 ± 4 yrs) and age‐matched males (n = 21; 24 ± 4 yrs). All the females completed the PLM assessment during the early (EF) and late follicular (LF) phase of their menstrual cycle, based on self‐reporting of menses. Lower limb microvascular function was assessed during PLM via changes in leg blood flow measured as peak change from baseline (LBFΔPEAK) and area‐under‐the‐curve (LBF AUC). Thigh volume, a known modulator of PLM responses, was also assessed in the passively moved limb in all participants via anthropometric measurements. Blood samples were obtained at rest from the antecubital vein of the arm to determine differences in E2 between the EF and LF phases of the menstrual cycle.ResultsThigh volume differed significantly between the females and age‐matched males (F: 4.9 ± 1.5 L; M: 8.2 ± 1.6 L; p &lt; 0.01) and, when examined in all participants, thigh volume was significantly correlated to PLM‐induced hyperemia evaluated as both LBFΔPEAK (r = 0.6; p &lt; 0.01) and LBF AUC (r = 0.4; p = 0.014). When the group differences in thigh volume were accounted for in the PLM analysis by employing thigh volume as a co‐variate, no significant differences in LBFΔPEAK (F: 542 ± 444; M: 794 ± 444 ml; p = 0.15) and LBF AUC (F: 164 ± 227; M: 249 ± 227 ml; p = 0.3) were revealed. The females reported no significant differences in PLM‐induced blood flow responses across menstrual cycle phases (EF vs LF) evaluated as LBFΔPEAK (EF: 385 ± 186; LF: 386 ± 154 ml; p = 0.9) or LBF AUC (EF: 123 ± 100; LF: 58 ± 84 ml; p = 0.11). Plasma E2 levels were significantly higher in females in the LF phase (215 ± 149 pg/ml) when compared to the EF phase (149 ± 98 pg/ml) of the menstrual cycle (p = 0.01).ConclusionThis study revealed no differences in PLM‐induced LBF responses in premenopausal females when compared to age‐matched males or when assessed across follicular menstrual cycle phases.