17β-estradiol and xenoestrogens reveal synergistic effect on mitochondria of human sperm

Objectives: The aim of the study was to investigate the influence of 17β-estradiol (main endogenous estrogen) and selected xenoestrogens (genistein, bisphenol-A), individually and in combination, on the mitochondrial function of human sper­matozoa. In natural environment, human beings are exposed to multiple xenoestrogens, so their impact is combined with endogenous steroids. Material and methods: The effects of ligands on human spermatozoa were assessed regarding the following phenomena: spermatozoa vitality (propidium iodide staining), phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein), mitochondrial membrane potential (using JC-1 fluorochrome), and production of superoxide anion in mitochondria (using MitoSOX RED dye). Results: Two-hour incubation of spermatozoa with 17β-estradiol, genistein, and bisphenol-A neither altered cell vitality nor stimulated phosphatidylserine membrane translocation. Incubation of spermatozoa with 17β-estradiol or bisphenol-A sepa­rately, as well as incubation with the three ligands simultaneously, resulted in altered mitochondrial membrane potential. Spermatozoa incubation with the three ligands significantly increased the mitochondrial superoxide anion level. Conclusions: It seems safe to conclude that human spermatozoa mitochondria are target cell structures for both, 17β-estradiol and xenoestrogens. The reaction to the 17β-estradiol and xenoestrogens mixture suggests a synergistic mechanism of action. Xenoestrogens may increase the sensitivity of spermatozoa to 17β-estradiol

Methylation of Estrogen Receptor 1 Gene in the Paraspinal Muscles of Girls with Idiopathic Scoliosis and Its Association with Disease Severity

Idiopathic scoliosis (IS) is a multifactorial disease with epigenetic modifications. Tissue dependent and differentially methylated regions (T-DMRs) may regulate tissue-specific expression of the estrogen receptor 1 gene (ESR1). This study aimed to analyze methylation levels within T-DMR1 and T-DMR2 and its concatenation with ESR1 expression of IS patients. The study involved 87 tissue samples (deep paravertebral muscles, both on the convex and the concave side of the curve, and from back superficial muscles) from 29 girls who underwent an operation due to IS. Patient subgroups were analyzed according to Cobb angle ≤70° vs. >70°. Methylation was significantly higher in the superficial muscles than in deep paravertebral muscles in half of the T-DMR1 CpGs and all T-DMR2 CpGs. The methylation level correlated with ESR1 expression level on the concave, but not convex, side of the curvature in a majority of the T-DMR2 CpGs. The T-DMR2 methylation level in the deep paravertebral muscles on the curvature’s concave side was significantly lower in patients with a Cobb angle ≤70° in four CpGs. DNA methylation of the T-DMRs is specific to muscle tissue location and may be related to ESR1 expression regulation. Additionally, the difference in T-DMR2 methylation may be associated with IS severity

Association of LBX1 Gene Methylation Level with Disease Severity in Patients with Idiopathic Scoliosis: Study on Deep Paravertebral Muscles

Idiopathic scoliosis (IS) is a multifactorial disease with a genetic background. The association of Ladybird Homeobox 1 (LBX1) polymorphisms with IS has been proven in multiple studies. However, the epigenetic mechanisms have not been evaluated. This study aimed to evaluate the LBX1 methylation level in deep paravertebral muscles in order to analyze its association with IS occurrence and/or IS severity. Fifty-seven IS patients and twenty non-IS patients were examined for the paravertebral muscles’ methylation level of the LBX1 promoter region. There was no significant difference in methylation level within paravertebral muscles between patients vs. controls, except for one CpG site. The comparison of the paravertebral muscles’ LBX1 promoter region methylation level between patients with a major curve angle of ≤70° vs. >70° revealed significantly higher methylation levels in 17 of 23 analyzed CpG sequences at the convex side of the curvature in patients with a major curve angle of >70° for the reverse strand promoter region. The association between LBX1 promoter methylation and IS severity was demonstrated. In patients with severe IS, the deep paravertebral muscles show an asymmetric LBX1 promoter region methylation level, higher at the convex scoliosis side, which reveals the role of locally acting factors in IS progression

MicroRNA expression profile analysis in human skeletal muscle tissue: Selection of critical reference

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs, that modulate gene expression by targeting messenger RNA of many processes. Thus, miRNAs are key regulators of both physiological and pathological settings. Reliable results of quantitative miRNA evaluation depend on suitable reference genes (RGs) for data normalization. To date, no consensus has been reached on the best RG for muscle tissue. We assessed RGs stability in skeletal muscle tissue in patients with spinal deformity. Ninety tissue samples were obtained from the deep paravertebral muscles from the convex and concave sides of the spinal curvature, as well as the superficial paraspinal muscles. We evaluated the stability of twelve miRNAs (hsa-miR-1–3p, hsa-miR-1–5p, hsa-miR-26b-5p, hsa-miR-92a-3p, hsa-miR-133a-3p, hsa-miR-133a-5p, hsa-miR-133b, hsa-miR-191–5p, hsa-miR-206, hsa-miR-208b-5p, hsa-miR-486–5p, hsa-miR-499a-5p), finding three to be indicative of reference miRNA, and nine as muscle-tissue specific. Stability was quantified using four statistical tools and a comprehensive ranking system. Three miRNAs were indicated as the most stable, and we assessed hsa-miR-486–5p as the most, and hsa-miR-208b-5p as the least suitable RGs for miRNA quantitative analyses. We recommend using a minimum of three RGs miRNA to normalize RT-qPCR data. Finally, qPCR efficiency should always be considered. To obtain consistent results, data normalization in muscle tissue is required

Hydroxyflutamide alters the characteristics of live boar spermatozoa

Our previous study revealed that in vitro incubation of boar ejaculates with hydroxyflutamide (OH-Flu) causes changes in sperm plasma membrane integrity and its stability and sperm mitochondrial oxidative capability. To broaden the knowledge of cellular physiology of spermatozoa, we investigated direct effects of OH-Flu administered for 2 and 24 hours at concentrations of 5, 50, and 100 µg/mL, on sperm mitochondrial membrane potential and mitochondrial superoxide anion production using JC-1 dye and MitoSOX Red fluorescent probe, respectively. We further measured phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the sperm plasma membrane using an annexin-V binding assay. To provide new information of direct effects of OH-Flu on cell signaling pathway, we measured sperm intracellular calcium ion dynamics using Fluo-3. Finally, we assessed sperm motility using a computer-assisted spermatozoa analysis system. Motile sperm were highlighted using the "C-Ruch" computer program for detailed analysis of the straight line velocity distribution. For each functional test, boar spermatozoa were examined and analyzed by flow cytometry and/or confocal microscopy. The results revealed a significant decrease (P < 0.05) in sperm mitochondrial membrane potential and a concomitant increase (P < 0.05) in mitochondrial superoxide anion production after a 2-hour incubation with 50 mu g OH-Flu compared with the respective controls and other doses used (P < 0.05). The adverse effects of OH-Flu become strengthened over time (P < 0.05). Notably, 50 and 100 µg OH-Flu appeared to be effective in decreasing sperm motility. Hydroxyflutamide significantly decreased (P < 0.05) the fast sperm subpopulation percentage after 15 minutes and reduced the straight line velocity distribution (P < 0.05). An assessment of PST revealed an increase in the percentage of PST-positive spermatozoa (P < 0.05)only after exposure to OH-Flu for 24 hours. Moreover, OH-Flu at all concentrations induced a rapid increase in sperm intracellular calcium ion concentration. Altogether, the altered in vitro characteristics of live boar spermatozoa provide new insight into direct effects of OH-Flu on sperm mitochondrial membrane potential, superoxide anion production, translocation of membrane phosphatidylserine, free calcium ion dynamics, and sperm motility

Differential Expression of HIF1A, EPAS1, and VEGF Genes in Benign and Malignant Ovarian Neoplasia

Ovarian cancer (OC) has the highest mortality rate of all gynecological malignancies. Moreover, at the time of the first clinical manifestation, most patients have an advanced stage of the disease. Our study examined differences in mRNA levels of hypoxia-inducible factor 1-alpha (HIF1A); endothelial PAS domain protein 1, also known as hypoxia-inducible factor 2-alpha (HIF2A/EPAS1); and vascular endothelial growth factor A (VEGFA) between cancerous tissue, benign hyperplastic changes in the ovary, and normal tissue. Our cohorts consisted of 52 patients diagnosed with OC (n = 55), benign non-cancerous changes (n = 21), and normal tissue samples (n = 38). The mRNA expression level was evaluated using RT-qPCR. We found that gene expression changes were visible not only in the case-control study, but also along with changes in severity. Additionally, the gene expression was differentiated in age, BMI, menopausal status, and the number of comorbidy-related groups. Furthermore, our findings demonstrate that analyzing the correlation between genes is essential. In a case-to-case and case-to-control study, we observed disturbances in the expression levels of interdependent genes. Our findings suggest that mutual association in the expression of both HIF1A and HIF2A/EPAS1 with VEGFA has prognostic importance for patients with OC. Our observations may help identify patients for clinical trials aimed at inhibiting the hypoxia-induced neovascularization-dependent pathways

Effect of Pentacyclic Triterpenoids-Rich Callus Extract of <i>Chaenomeles japonica</i> (Thunb.) Lindl. ex Spach on Viability, Morphology, and Proliferation of Normal Human Skin Fibroblasts

The effect of the well-characterized callus extract of Chaenomeles japonica on viability, morphology, and proliferation of normal human skin fibroblasts was investigated. The phytochemical analysis was performed using the ultra-high performance liquid chromatography method. The total phenolic, phenolic acid, and flavonoid contents were determined spectrophotometrically. The antioxidant activity was investigated using the DPPH (1,1-Diphenyl-1-picrylhydrazyl Radical Scavenging), FRAP (Ferric Reducing Antioxidant Power), and CUPRAC (CUPric Reducing Antioxidant Capacity) assays. The callus growth index during passages was high as well as the content of pentacyclic triterpenoids. The microscopic observations of the fibroblast viability, morphology and the evaluation of the proliferation ratio (xCELLigence system) proved that the influence of callus extract on the fibroblasts was dose-dependent. The evaluated level of fibroblasts proliferation rate after 72 h of incubation with callus extract at concentration 12.5 &#181;g L&#8722;1 was the highest compared to all the analyzed ligands. Moreover, callus extract administrated for 72 h caused a significant increase in the proliferation rate in comparison with the control group (5.7 &#177; 0.1 vs. 4.4 &#177; 0.9; p &lt; 0.01). The preliminary studies carried out may suggest that the callus extract rich in triterpenoids may be a potential source of cosmetic ingredients with a beneficial effect on human skin

Estrogen Receptor Type 1 and Type 2 Presence in Paravertebral Skeletal Muscles: Expression Level and Relation to Phenotype in Children with Idiopathic Scoliosis

The study aimed to detect the presence and assess the expression levels of the estrogen receptors type 1 (ESR1) and type 2 (ESR2) within paravertebral skeletal muscles of female patients with idiopathic scoliosis (IS) in relation to phenotype parameters. Intraoperatively, the muscle samples were obtained from 35 adolescent females. The RT-qPCR, western blot and immunohistochemistry techniques were applied. The ESR1 and ESR2 were detected within paravertebral skeletal muscle cells, either the superficial or the deep ones. The ESR1 expression level was significantly higher in the deep muscles compared to the superficial ones. A left-right asymmetry of the ESR1 and ESR2 expression level was demonstrated in the deep muscles. There was a significant relationship between the expression asymmetry and either the Cobb angle or the progression risk factor: both parameters decreased to the smallest values in the case of symmetric ESR1 or ESR2 expression, while they increased with increasing expression asymmetry. In conclusion, the ESR1 and ESR2 presence was confirmed in skeletal paravertebral muscles of patients with idiopathic scoliosis. The increased expression level and asymmetry of estrogen receptors in deep skeletal muscles was related to increasing scoliotic deformity magnitude or increasing risk of deformity deterioration. These findings may highlight the etiopathogenesis of IS in children

Impact of Biometric Patient Data, Probiotic Supplementation, and Selected Gut Microorganisms on Calprotectin, Zonulin, and sIgA Concentrations in the Stool of Adults Aged 18&ndash;74 Years

Alterations to the intestinal barrier may be involved in the pathogenesis of various chronic diseases. The diagnosis of mucosal barrier disruption has become a new therapeutic target for disease prevention. The aim of this study was to determine whether various patient demographic and biometric data, often not included in diagnostic analyses, may affect calprotectin, zonulin, and sIgA biomarker values. Stool markers&rsquo; levels in 160 samples were measured colorimetrically. The analysis of twenty key bacteria (15 genera and 5 species) was carried out on the basis of diagnostic tests, including cultures and molecular tests. The concentrations of selected markers were within reference ranges for most patients. The sIgA level was significantly lower in participants declaring probiotics supplementation (p = 0.0464). We did not observe differences in gastrointestinal discomfort in participants. We found significant differences in the sIgA level between the 29&ndash;55 years and &gt;55 years age-related intervals groups (p = 0.0191), together with a significant decreasing trend (p = 0.0337) in age-dependent sIgA concentration. We observed complex interdependencies and relationships between their microbiota and the analyzed biomarkers. For correct clinical application, standardized values of calprotectin and sIgA should be determined, especially in elderly patients. We observed a correlation between the composition of the gut community and biomarker levels, although it requires further in-depth analysis