The influence of retinol concentration in liquid crystal formula on epidermal growth factor, interleukin-6 and transglutaminase-1 mRNA expression in the epidermis.

Topical retinol application effectively reduces the effects of photoaging and improves skin condition, e.g. influences the process of keratinization of the epidermis, which improves stratum corneum structure and reduces transepidermal water loss. However, cosmetics use lower concentrations of retinol, which has been associated with emerging hypersensitivity reactions as well as redness and irritation of the skin. The question arises whether the vehicle used in the cosmetic may be important in stimulating the inflammatory reaction in the skin and if the concentration of retinol used could significantly affects the growth of epidermal cells. The aim of this study was to evaluate the effects of different concentrations of liquid crystal retinol (0.15%, 0.3% and 0.5%) on the clinical and histological characteristics of a reconstructed epidermis skin model. It also compares the effectiveness of 0.3% retinol formula in liquid crystal to that in lipid. The study used reconstructed human epidermis tissue containing normal human keratinocytes. Four original formulas containing retinol were tested: 0.15%, 0.3% and 0.5% with a liquid crystal base, and 0.3% with a lipid base. Interleukin 6 (IL-6), transglutaminase-1 (TGM1), and epidermal growth factor (EGF) mRNA expression was measured expression of the skin-equivalent tissue for 10 days of exposure. Histopathological analysis and mRNA quantification were performed. Gene expression was analyzed by total mRNA extraction. All liquid crystal formulas induced higher EGF mRNA expression than lipid base formula. IL-6 expression did not differ significantly from the DPBS reference values. Interestingly, TGM1 expression was found to increase together with increasing retinol concentration (0.15%, 0.3%, 0.5%). Histological examination revealed changes in epidermal structure, mainly hyperkeratinization of the stratum corneum. Our results support the hypothesis liquid crystal formula might be regarded as more beneficial since it inducess less pro-inflammatory action manifested by lowered expression IL-6. In addition, EGF expression was found to correlate significantly with the retinol concentration of the liquid crystal formula: 0.5% > 0.3% > 0.15% (P < 0.05). Lower concentrations may increase TGM1 expression, thus enhancing the formation of a protective layer of cornified envelope.

Psychiatric and neurocognitive consequences of endogenous hypercortisolism.

Psychiatric and neurocognitive symptoms due to hypercortisolism were already described by Harvey Cushing in his original paper on patients with Cushing's syndrome (CS). Nowadays, it is well known that psychiatric and cognitive complaints are two of the most common, and most distressing, symptoms in patients with CS. Psychiatric symptoms are indeed a major clinical manifestation of CS. The most commonly observed psychiatric conditions are depression and anxiety, whilst mania and psychosis are less common. Several domains of cognitive function are impaired at diagnosis, including episodic and working memory, executive function and attention. Following treatment, one-fourth of the patients still experience depressed mood, and the cognitive impairments are only partially restored. Consequently, quality of life in patients with CS is severely and persistently affected. Neuroimaging studies have also illustrated the deleterious effects of hypercortisolism on the brain by demonstrating reduced grey matter volumes and cortical thickness, altered resting-state functional responses and during cognitive tasks, as well as widespread reduced white matter integrity, especially in structures important for cognitive function and emotional processing, both before and after successful abrogation of hypercortisolism. In this paper, we summarize the current knowledge on the psychiatric and neurocognitive consequences of hypercortisolism in patients with CS, both before, and after successful treatment. In addition, we review the structural and functional brain abnormalities associated with hypercortisolism and discuss the influence of these factors on quality of life.

Photoswitchable transition metal complexes with azobenzene-functionalized imine-based ligands: structural and kinetic analysis.

We report on the characterization of two imine type ligands containing photoresponsive azobenzene units as side groups and their transition metal ions complexes. The ligands, both free and in their complexes undergo trans- > cis photoisomerization after irradiation with UV light, but binding of metal ions reduces both the photoisomerisation reaction rates and cis isomer concentrations in the photostationary states. The greatest diminution in the photoisomerisation rate was observed for the complex containing Cd(ii), the heaviest among the various transition metal ions tested in this study.

Bacterial Contamination of Boar Semen and its Relationship to Sperm Quality Preserved in Commercial Extender Containing Gentamicin Sulfate.

This study was designed to determine the degree and type of bacterial contamination in boar semen (79 ejaculates from Large White and Landrace boars) and its consequences for sperm quality during storage (27 extended semen samples, 16°C for five days) under practical conditions of artificial insemination (AI). The results revealed the presence of aerobic bacteria in 99% of the ejaculates (from 80 to 370 ×106 colony-forming units/mL). Most of the ejaculates contained two or three bacterial contaminants, while the Staphylococcus, Streptococcus, and Pseudomonas bacterial genera were most frequently isolated. Also detected were Enterobacter spp., Bacillus spp., Proteus spp., Escherichia coli, P. fluorescens, and P. aeruginosa. In general, the growth of certain bacterial types isolated prior to semen processing (Enterobacter spp., E. coli, P. fluorescens, and P. aeruginosa) was not discovered on different days of storage, but fluctuations (with a tendency towards increases) were found in the frequencies of Bacillus spp., Pseudomonas spp., and Staphylococcus spp. isolates up to the end of storage. Semen preserved for five days exhibited decreases in sperm motility and increases in the average number of total aerobic bacteria; this was associated with sperm agglutination, plasma membrane disruption, and acrosome damage. We inferred that, due to the different degrees and types of bacterial contaminants in the boar ejaculates, the inhibitory activity of some antimicrobial agents used in swine extenders (such as gentamicin sulfate) may be limited. Because such agents can contribute to the overgrowth of certain aerobic bacteria and a reduction in the quality of stored semen, procedures with high standards of hygiene and microbiological control should be used when processing boar semen.

Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus.

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.

Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 µg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

Evaluation of sperm genomic integrity of normozoospermic men: a prospective study.

The objective of our study was to evaluate the incidence of spermatozoa with nuclear DNA strand breaks in patients with normal routine sperm parameters (26 subjects). Sperm DNA fragmentation was measured using TUNEL test assessed in flow cytometer. Variable percentages of sperm with damaged DNA (9.42 +/- 7.68%; range: 2-36) were found. Two categories of patients were distinguished: (1) patients (8 out of 26 subjects) with < or = 4% of TUNEL-positive sperm and (2) patients (18 out of 26 subjects) with > 4% of TUNEL-positive sperm. A significantly lower percentage of normal sperm forms was found in patients with > 4% of TUNEL-positive sperm than in patients with < or = 4% of TUNEL-positive sperm. Moreover, a significant negative correlation (r(s) = -0.50) was noted only between a proportion of normal sperm forms and a proportion of TUNEL-positive spermatozoa. In electron microscope, a large number of spermatozoa with immature chromatin was observed more frequently in subjects with > 4% of TUNEL-positive cells (11 out of 18 subjects). Our results suggest that in some patients with normal routine sperm parameters, DNA fragmentation may be associated with poor sperm morphology. The diminished sperm genomic integrity may result from molecular disturbances in nuclear remodeling process during spermiogenesis. TUNEL assay is a screening tool that may help to discriminate between fertile and infertile men and may help to predict successful in vitro fertilization.

Diagnostic evaluation of oxidoreductive capability of sperm mitochondria.

In the present paper, morphological and functional features of human sperm midpiece, contributing to the assessment of sperm fertility potential, have been described. The NADH-dependent NBT screening assay was used to identify and visualise: 1/ morphological defects of sperm midpiece, 2/ immature sperm forms with extensive cytoplasmic retention, reflecting developmental failure in spermatogenic remodelling process, 3/ cytoplasmic sperm conglomerates, related to apoptotic bodies and 4/ sperm NADH-dependent oxidoreductase system at the mitochondrial level, related to the reaction intensity. The used assay is an adequate marker of sperm mitochondrial activity and sperm maturity. It can also help discover sperm defects that result in asthenozoospermia and can be used as an additional indicator in the evaluation of the sperm midpiece, as well as in routine morphological examination of spermatozoa, having a considerable predictive value for in vivo and in vitro fertilization.

Aging process of epithelial cells of the rat prostate lateral lobe in experimental hyperprolactinemia induced by haloperidol.

The aim of the study was to examine the influence of hyperprolactinemia, induced by haloperidol (HAL) on age related morphology and function changes of epithelial cells in rat prostate lateral lobe. The study was performed on sexually mature male rats. Serum concentrations of prolactin (PRL) and testosterone (T) were measured. Tissue sections were evaluated with light and electron microscopy. Immunohistochemical reactions for Anti-Proliferating Cell Nuclear Antigen (PCNA) were performed. In rats of the experimental group, the mean concentration of: PRL was more than twice higher, whereas T concentration was almost twice lower than that in the control group. Light microscopy visualized the following: hypertrophy and epithelium hyperplasia of the glandular ducts, associated with increased PCNA expression. Electron microscopy revealed changes in columnar epithelial cells, concerning organelles, engaged in protein synthesis and secretion.

Germ cells with nuclear DNA fragmentation related to apoptotic cells in rat testis in experimental hyperprolactinemia induced by metoclopramide.

The cells with nuclear DNA fragmentation related to apoptosis were detected by TUNEL technique in the seminiferous epithelium of control rats and of rats with experimental hyperprolactinemia induced by metoclopramide. The percentage of convoluted tubules with apoptotic cells and the number of apoptotic cells (predominantly spermatogonia and spermatocytes) was increased in the experimental group. The results indicated stage-specific germ cell apoptosis. In the experimental group, apoptotic cells were most evident at early (I-IV), middle (VII-VIII) and late (XII-XIV) stages of the seminiferous epithelium cycle, as revealed by light and electron microscopy. We suggest that a decreased concentration of testosterone and an increased concentration of prolactin could disturb spermatogenesis and contribute to the intensive apoptosis of germ cells in rats with hyperprolactinemia. Sertoli cells which have receptors for testosterone and prolactin and play an important role in spermatogenesis and in the initiation of apoptosis in seminiferous epithelium, could mediate such an influence of both hormones.

Computerized analysis of cytochemical reactions for dehydrogenases and oxygraphic studies as methods to evaluate the function of the mitochondrial sheath in rat spermatozoa.

Cytochemical reactions for mitochondrial NADH-dependent dehydrogenases (diaphorase/NADH which is related to flavoprotein), NAD-dependent dehydrogenases (isocitrate, malate) and succinate dehydrogenase were carried out in rat spermatozoa. In addition to a morphological evaluation, the intensity of the reactions was assessed using a computer image analysing system (Quantimet 600 S). The intensity of the reactions was examined in sperm midpieces by measuring integrated optical density (IOD) and mean optical density (MOD). The activity of mitochondrial respiratory chain complexes was also analysed using the polarographic method. In the population of spermatozoa studied, all whole spermatozoa midpieces were completely filled with formazans, the product of the cytochemical reaction. These morphological findings corresponded to the values obtained for IOD and MOD for the given enzymes. In the oxygraphic studies, the spermatozoa demonstrated consumption of oxygen in the presence of substrates for I, II and IV complexes and their mitochondria revealed normal integrity and sensitivity to the substrates and inhibitors. However, the oxygraphic studies revealed differences between the sperm and somatic cells. These differences concerned the stimulation of pyruvate oxidation by malate, the lack of an effect of malonic acid on phenazine methosulphate (an acceptor of electrons) oxidation and the lack of an effect of cytochrome c on ascorbate oxidation. The cytochemical method, together with densitometric measurements, enables: (1) the reaction intensity to be determined objectively; (2) subtle and dramatic differences in reaction intensity to be revealed between spermatozoa that do not differ under morphological evaluation of the intensity; (3) possible defects within the mitochondrial sheath to be located and assessed in a large number of spermatozoa. This method can be used as a screening method alongside the routine morphological examination of spermatozoa. On the other hand, the oxygraphic method in the inner membrane of mitochondria can reveal functional changes which are related to the action of respiratory chain complexes and display characteristic features of mitochondria energy metabolism. The methods used are complementary and allow the complex evaluation of mitochondria in spermatozoa. Both methods can be used in experimental and clinical studies.

[Internal standardization of sperm morphology evaluated according to strict morphological criteria]. / Próba standaryzacji badan morfologicznych plemników w oparciu o material wlasny pracowni seminologicznej.

OBJECTIVE: To evaluate sperm morphology and find cut-off values for local andrology lab based on morphological strict criteria. To compare the results to WHO guidelines. MATERIAL AND METHODS: Strict morphological criteria were applied to 300 sperm smears stained according to the Papanicolaou method. Specific sperm defects were described in details. The results were analyzed statistically. RESULTS: The normal sperm morphology was found in 18.54% of cases, which is less than the cut-off value suggested in the WHO guidelines. The lab cut-off value aimed by the 25th percentile was 8%. CONCLUSION: Sperm morphology requires broader multi-center standardization to enable compatible exchange of morphological data and find predictive factors of sperm fertilizing potential.

[Changes in sperm mitochondria in patients with asthenozoospermia]. / Zmiany we wstawkach plemników pacjentów z astenozoospermia.

OBJECTIVE: To evaluate functional and ultrastructural alterations of the spermatozoa midpieces in patients with asthenozoospermia and to find a correlation between the damage of the midpieces and loss of sperm motility. MATERIAL AND METHODS: Routine, morphological assessment of the midpieces stained according to the Papanicolaou method, cytochemical study of the mitochondrial activity using reaction for the diaphorase/ NADH according to the Piasecka method and electron-microscopic investigation of the midpiece structures were performed. RESULTS: The cytochemical reaction for diaphorase/NADH revealed disorders of the mitochondrial activity and subtle and drastic malformations in the spermatozoa midpieces. The unusually thickened midpieces contained the supernumerary mitochondria. In patients with severe asthenozoospermia, the damage of the accessory fibres and axonemal complex located in the midpiece, were obtained also. CONCLUSION: This study indicates that mitochondrial defects are one of the causes that may account for loss of sperm motility in the population of patients.

Phospholipid content and lamellar structures in the epididymal epithelial cells of rats treated chronically with lead acetate [Pb(II)].

The electron-microscopic observations accomplished covered epididymal epithelial cells of rats receiving lead acetate for five times longer than the duration of one spermatogenesis. These cells were found to possess a large number of vacuoles and conglomerates containing plicated membranes or tightly packed myelin-like lamellar formations. Further observations also revealed the formation of lamellar structures in mitochondria, dilatation of cisternae in the Golgi apparatus, and increased phagocytosis of spermatozoa by epithelial cells. The presence of a large amount of membranous material correlated with the increased content of phospholipids in epididymal epithelial cells. It may be suggested that the presence of such a great quantity of lamellar structures in epididymal epithelial cells of rats treated chronically with lead is the result of several processes, including the augmented synthesis of membranes associated with encircling the deposits of lead, autophagy in the cells, as well as intensified phagocytosis of spermatozoa.

The ultrastructure of the testis in rats after long-term treatment with lead acetate.

Studies were performed to investigate the influence of long-term lead acetate treatment on morphology of rat testis. No marked changes were observed by means of light microscopy. At all stages (I-XIV) of the seminiferous epithelium cycle, all generations and layers of spermatogenic cells were present. Electron-microscopic studies did not reveal any ultrastructural changes neither in seminiferous epithelium nor in Sertoli cells. In Leydig cells also, no ultra-structural abnormalities were visible. Macrophages of testicular interstitial tissue contained electron-dense inclusions, usually located inside phagolisosome-like vacuoles. X-ray micro-analysis revealed that the inclusions contained lead.

Ultrastructure of spermatozoa from the cauda epididymis in rat chronically treated with lead acetate [Pb(II)].

Electron microscopic studies were performed on spermatozoa from the lumen of cauda epididymis in rats receiving lead acetate (II) for a period 5-fold longer than one spermatogenesis. There were numerous ultrastructural changes of spermatozoa. Mitochondrial spiral, outer dense fibers and axoneme were affected. Based on the present results it may be suggested that lead can damage spermatozoa in the epididymis.