Here we investigate the stress-signaling responsible for the effects of acute/repeated psychological stresses (the most common stresses in human society) on spermatozoa number and functionality, as ...well as the transcriptional profile of mitochondrial dynamics markers by using the in vivo and ex vivo approaches. Acute and repeated stress inhibit spermatozoa functionality (acute -> 3.2-fold, repeated -> 2.5-fold), while only repeated stress reduces the spermatozoa number (1.7-fold). Stress hormones mimic these effects and decrease the spermatozoa functionality (adrenaline: 10 µM -> 2.4-fold, 100 µM - > 2.8-fold; hydrocortisone: 50 pM -> 2.7-fold, 500 pM -> 8.5-fold). They also significantly disturb the transcriptional profile of all main mitochondrial dynamics markers in spermatozoa. Ex vivo manipulation of stress signaling in spermatozoa reveals that most of these effects are mediated through ɑ1-and/or-β-adrenergic receptors. The transcription of these receptors and their kinases in the same samples is under the significant influence of adrenergic signaling. Our results are the first to show the importance of mitochondrial dynamics markers in spermatozoa since the transcriptional profiles of sixteen-out-of-ninteen are disturbed by manipulation of stress-hormones-signaling. This is a completely new molecular approach to assess spermatozoa functionality and it is important for a better understanding of the correlations between stress, environmental-life-style and other factors, and male (in)fertility.
Here, we study possible mechanisms of (in/sub)fertility related to the acute or repeated psychological stresses (the most common stresses in human society) by following the transcriptional profile of ...22 mitochondrial dynamics/function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality. An in vivo study mimicking acute (once for 3 h) and repeated (3 h for 10 consecutive days) psychophysical stress was performed on adult rats. The analysis of hormones, the number/functionality of spermatozoa, and 44 transcriptional markers were performed on individual samples from up to 12 animals per group. Results showed that both types of stress reduced spermatozoa functionality (acute by 4.4-fold, repeated by 3.3-fold) and ATP production (acute by 2.3-fold, repeated by 14.5-fold), while only repeated stress reduces the number of spermatozoa (1.9-fold). Stress significantly disturbed transcription of 34-out-of-44 markers (77%). Mitochondrial dynamics and functionality markers: 18-out-of-22 =>82% (mitochondrial-biogenesis-markers –>6-out-of-8 =>75%; mitochondrial-fusion-markers –>3-out-of-3 =>100%; mitochondrial-fission-markers –>1-out-of-2 =>50%; mitochondrial-autophagy-markers –>3-out-of-3 =>100%; mitochondrial-functionality-markers –>5-out-of-6 =>83%). Markers of signaling pathways regulating both mitochondrial dynamics/functionality and spermatozoa number/functionality important for male (in/sub)fertility –>16-out-of-22 =>73% (cAMP-signaling-markers –>8-out-of-12 =>67%; MAPK-signaling-markers –>8-out-of-10 =>80%). Accordingly, stress-triggered changes of transcriptional profile of mitochondrial dynamics/functionality markers as well as signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality represent adaptive mechanisms.
The present work was undertaken to determine the total phenol, flavonoid and tannin content in five extracts of Origanum vulgare growing wild in southwest Serbia as well as to evaluate antioxidant ...and antimicrobial activity towards 29 strains of human pathogenic and food spoilage bacteria and fungi. In order to define a chemotype of this O. vulgare, chemical composition of essential oil was determined. The major compounds were: sabinene, terpinen-4-ol, 1.8 cineole, γ-terpinene and caryophyllene oxide. The concentrations of total phenols were examined using Folin–Ciocalteu reagent and the obtained values ranged from 84.5 to 235 mg GA/g. By using aluminium chloride method, the concentrations of flavonoids were obtained and the values ranged from 57.1 to 132 mg RU/g. In determining the amount of tannins, the method with butanol–HCl reagent was used and the obtained values were between 1.25 and 4.02 mg CCh/g. Antioxidant activity was evaluated by measuring the scavenging capacity of extracts on DPPH. Tested extracts expressed strong scavenger activity with IC50 values between 34.5 and 86 μg/ml. The ethanol extract was the most active. The strongest antibacterial activity, determined by microdilution method, was detected on G+ bacteria such as Bacillus species and Staphylococcus aureus, obtaining minimal inhibitory concentration (MIC) 0.16 mg/ml while antifungal activity was moderate with MICs between 2.5 and 20 mg/mL.
•We determined chemical composition of essential oils.•We examined five different extracts of aerial parts from oregano.•All extracts showed significant antimicrobial effect.•Good antioxidative activity.
Aim
A growing body of evidence pointed correlation between insulin‐resistance, testosterone level and infertility, but there is scarce information about mechanisms. The aim of this study was to ...identify the possible mechanism linking the insulin‐resistance with testosterone‐producing‐Leydig‐cells functionality.
Methods
We applied in vivo and in vitro approaches. The in vivo model of functional genomics is represented by INSR/IGF1R‐deficient‐testosterone‐producing Leydig cells obtained from the prepubertal (P21) and adult (P80) male mice with insulin + IGF1‐receptors deletion in steroidogenic cells (Insr/Igf1r‐DKO). The in vitro model of INSR/IGF1R‐deficient‐cell was mimicked by blockade of insulin/IGF1‐receptors on the primary culture of P21 and P80 Leydig cells.
Results
Leydig‐cell‐specific‐insulin‐resistance induce the development of estrogenic characteristics of progenitor Leydig cells in prepubertal mice and mature Leydig cells in adult mice, followed with a dramatic reduction of androgen phenotype. Level of androgens in serum, testes and Leydig cells decrease as a consequence of the dramatic reduction of steroidogenic capacity and activity as well as all functional markers of Leydig cell. Oppositely, the markers for female‐steroidogenic‐cell differentiation and function increase. The physiological significances are the higher level of testosterone‐to‐estradiol‐conversion in double‐knock‐out‐mice of both ages and few spermatozoa in adults. Intriguingly, the transcription of pro‐male sexual differentiation markers Sry/Sox9 increased in P21‐Leydig‐cells, questioning the current view about the antagonistic genetic programs underlying gonadal sex determination.
Conclusion
The results provide new molecular mechanisms leading to the development of the female phenotype in Leydig cells from Insr/Igf1r‐DKO mice and could help to better understand the correlation between insulin resistance, testosterone and male (in)fertility.
In the search for the possible role of the mitochondrial dynamics markers in spermatozoa adaptation, an in vivo approach was designed to mimic situations in which human populations are exposed to 3 h ...of repeated psychological stress (the most common stress in human society) at different time points during the day (24 h). The hormones (stress hormone corticosterone and testosterone), the number and the functionality of spermatozoa (response to acrosome-reaction-inducer progesterone), as well as the transcriptional profiles of 22 mitochondrial dynamics and function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality were followed at three time points (ZT3, ZT11, and ZT23). The results show that repeated stress significantly decreased the number and functionality of spermatozoa at all time points. In the same samples, the transcriptional profiles of 91% (20/22) of mitochondrial dynamics and functionality markers and 86% (19/22) of signaling molecules were disturbed after repeated stress. It is important to point out that similar molecular changes in transcriptional profiles were observed at ZT3 and ZT23, but the opposite was observed at ZT11, suggesting the circadian nature of the adaptive response. The results of PCA analysis show the significant separation of repeated stress effects during the inactive/light and active/dark phases of the day, suggesting the circadian timing of molecular adaptations.
Although age-related hypofunction of Leydig cells is well illustrated across species, its circadian nature has not been analyzed. Here we describe changes in circadian behavior in Leydig cells ...isolated from adult (3-month) and aged (18- and 24-month) rats. The results showed reduced circadian pattern of testosterone secretion in both groups of aged rats despite unchanged LH circadian secretion. Although arrhythmic, the expression of Insl3, another secretory product of Leydig cells, was decreased in both groups. Intracellular cAMP and most important steroidogenic genes (Star, Cyp11a1 and Cyp17a1), together with positive steroidogenic regulator (Nur77), showed preserved circadian rhythm in aging although rhythm robustness and expression level were attenuated in both aged groups. Aging compromised cholesterol mobilization and uptake by Leydig cells: the oscillatory transcription pattern of genes encoding HDL-receptor (Scarb1), hormone sensitive lipase (Lipe, enzyme that converts cholesterol esters from lipid droplets into free cholesterol) and protein responsible for forming the cholesterol esters (Soat2) were flattened in 24-month group. The majority of examined clock genes displayed circadian behavior in expression but only a few of them (Bmal1, Per1, Per2, Per3 and Rev-Erba) were reduced in 24-month-old group. Furthermore, aging reduced oscillatory expression pattern of Sirt1 and Nampt, genes encoding key enzymes that connect cellular metabolism and circadian network. Altogether circadian amplitude of Leydig cell's endocrine function decreased during aging. The results suggest that clock genes are more resistant to aging than genes involved in steroidogenesis supporting the hypothesis about peripheral clock involvement in rhythm maintenance during aging.
•Aging lowered circadian serum testosterone variations.•Aging decline circadian expression of key steroidogenic and clock genes.•Cholesterol metabolism and uptake are dampened in Leydig cells from aged rats.•During aging the changes in steroidogenesis precede changes in clock network.•Reduced circadian expression of Nampt/Sirt1 pointed decreased cellular metabolism.
In this study, we determined the concentration of total phenols, flavonoids, tannins, and proanthocyanidins in the water, diethyl ether, acetone, and ethanol extracts of Agrimonia eupatoria L. We ...also investigated the antioxidant activity of these extracts using two methods 2,2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power and their in vitro antimicrobial (antibacterial and antifungal) activity on some selected species of bacteria and fungi. In addition, the effects of the acetone and water extracts on the inhibition of biofilm formation of Proteus mirabilis and Pseudomonas aeruginosa were investigated using the crystal violet method. The concentration of total phenols was measured according to the Folin–Ciocalteu method and the values obtained ranged from 19.61 mgGA/g to 220.31 mgGA/g. The concentration of flavonoids was examined by the aluminum chloride method and the values obtained ranged from 20.58 mgRU/g to 97.06 mgRU/g. The total tannins concentration was measured by the polyvinylpolypyrrolidone method and the values obtained ranged from 3.06 mgGA/g to 207.27 mgGA/g. The concentration of proanthocyanidins was determined by the butanol–HCl method and the values obtained ranged from 4.15 CChE/g to 103.72 CChE/g. Among the various extracts studied, the acetone extract exhibited good antioxidant activity (97.13%, as determined by the DPPH method). The acetone extract was active in the absorbance value range from 2.2665 to 0.2495 (as determined by the reducing power method). The strongest antimicrobial activity was detected on G+ bacteria, especially on probiotic species, and the acetone extract demonstrated the highest activity. Biofilm inhibitory concentration required to reduce biofilm coverage by 50% values for acetone extract was 4315 μg/mL for P. mirabilis and 4469.5 μg/mL for P. aeruginosa. The results provide a basis for further research of this plant species.
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This study was designed to search for the possible mechanism(s) of male (in/sub)fertility by following the molecular response of spermatozoa on acute psychological stress (the most common stress in ...human society) and on a 20-h time-dependent recovery period. To mimic
in vivo
acute stress, the rats were exposed to immobilization once every 3 h. The recovery periods were as follows: 0 (immediately after stress and 3 h after the light is on—ZT3), 8 (ZT11), 14 (ZT17), and 20 (ZT23) h after stress. Results showed that acute stress provoked effects evident 20 h after the end of the stress period. Numbers of spermatozoa declined at ZT17 and ZT23, while functionality decreased at ZT3 and ZT11, but recovered at ZT17 and ZT23. Transcriptional profiles of 91% (20/22) of tracked mitochondrial dynamics and functionality markers and 91% (20/22) of signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality were disturbed after acute stress and during the recovery period. Most of the changes presented as increased transcription or protein expression at ZT23. The results of the principal component analysis (PCA) showed the clear separation of acute stress recovery effects during active/dark and inactive/light phases. The physiological relevance of these results is the recovered positive-acrosome-reaction, suggesting that molecular events are an adaptive mechanism, regulated by acute stress response signaling. The results of the PCA confirmed the separation of the effects of acute stress recovery on gene expression related to mitochondrial dynamics, cAMP, and MAPK signaling. The transcriptional patterns were different during the active and inactive phases. Most of the transcripts were highly expressed during the active phase, which is expected given that stress occurred at the beginning of the inactive phase. To the best of our knowledge, our results provide a completely new view and the first presentation of the markers of mitochondrial dynamics network in spermatozoa and their correlation with signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality during recovery from acute stress. Moreover, the interactions between the proteins important for spermatozoa homeostasis and functionality (MFN2 and PRKA catalytic subunit, MFN2 and p38MAPK) are shown for the first time. Since the existing literature suggests the importance of semen quality and male fertility not only as the fundamental marker of reproductive health but also as the fundamental biomarkers of overall health and harbingers for the development of comorbidity and mortality, we anticipate our result to be a starting point for more investigations considering the mitochondrial dynamics markers or their transcriptional profiles as possible predictors of (in/sub)fertility.
Controlled changes in mitochondrial biogenesis and morphology are required for cell survival and homeostasis, but the molecular mechanisms are largely unknown. Here, male and female prepubertal mice ...(P21) with insulin and IGF1 receptors deletions in steroidogenic tissues (Insr/Igf1r-DKO) were used to investigate transcription of the key regulators of mitochondrial biogenesis (Ppargc1a, Ppargc1b, Pparg, Nrf1, Tfam) and architecture in Leydig cells, ovaries, and adrenals. Results showed that the expression of PGC1, a master regulator of mitochondrial biogenesis and integrator of environmental signals, and its downstream target Tfam, significantly decreased in androgen-producing Leydig cells. This is followed by reduction of Mtnd1, a mitochondrial DNA encoded transcript whose core subunit belongs to the minimal assembly required for catalysis. The same markers remained unchanged in ovaries. In contrast, in adrenals, the pattern of transcripts for mitochondrial biogenesis markers was the same in both sexes, but opposite from that observed in Leydig cells. The level of transcripts for markers of mitochondrial architecture (Mfn1, Mfn2) significantly increased in Leydig cells from Insr/Igf1r-DKO, but not in ovaries. This was followed by mitochondrial morphology disturbance, suggesting that the mitochondrial phase of steroidogenesis could be affected. Indeed, basal and pregnenolone stimulated progesterone productions in the mitochondria of Leydig cells from Insr/Igf1r-DKO decreased more than androgen production, and were barely detectable. Our results are the first to show that INSR/IGF1R are important for mitochondrial biogenesis in gonadal steroidogenic cells of prepubertal males, but not females and they serve as important regulators of mitochondrial architecture and biogenesis markers in Leydig cells. Summary Sentence Insulin/IGF1 signaling is essential for expression of the main markers of mitochondrial biogenesis in steroidogenic cells of the testes, but not ovaries of prepubertal mice.
Patients are faced with multiple alternatives when selecting the preferred method for colorectal cancer screening, and there are multiple criteria to be considered in the decision-making process. We ...model patients’ choices using a multicriteria decision model, specifically an Analytic Hierarchy Process-based model, and propose a new approach for characterizing the idiosyncratic preference regions for individual patients. The new approach involves the development of a personalized sensitivity and stability analysis of preferences that provides pertinent insights about the changes that occur in a patient’s preferences, as he/she learns additional relevant medical information. We show how the insights derived from the sensitivity and stability of patients’ preferences could be used by a healthcare provider within the medical decision-making process.
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